Tat-derived transport polypeptides and fusion proteins

ABSTRACT

This invention relates to delivery of biologically active cargo molecules, such as polypeptides and nucleic acids, into the cytoplasm and nuclei of cells in vitro and in vivo. Intracellular delivery of cargo molecules according to this invention is accomplished by the use of novel transport polypeptides which comprise HIV tat protein or one or more portions thereof, and which are covalently attached to cargo molecules. The transport polypeptides in preferred embodiments of this invention are characterized by the presence of the tat basic region (amino acids 49-57), the absence of the tat cysteine-rich region (amino acids 22-36) and the absence of the tat exon 2-encoded carboxy-terminal domain (amino acids 73-86) of the naturally-occurring tat protein. By virtue of the absence of the cysteine-rich region, the preferred transport polypeptides of this invention solve the potential problems of spurious trans-activation and disulfide aggregation. The reduced size of the preferred transport polypeptides of this invention also minimizes interference with the biological activity of the cargo molecule.

This application is a continuation of application Ser. No. 08/235,403,filed Apr. 28, 1994, which is a continuation-in-part of application Ser.No. 08/158,015, filed Nov. 24, 1993, now abandoned, which is a filewrapper continuation of Ser. No. 07/636,662, filed Jan. 2, 1991, nowabandoned, which is a continuation-in-part of 07/454,450, filed Dec. 21,1989, now abandoned. Prior application Ser. No. 08/235,403 also claimspriority under 35 U.S.C. §120 from PCT application PCT/US93/07833, filedAug. 19, 1993, designating the United States and claiming priority fromSer. No. 07/934,375, filed Aug. 21, 1992, now abandoned.

Work described herein was supported, in part, by the National Institutesof Health, Whitehead Institute for Biomedical Research, Howard HughesMedical Institute and Johns Hopkins University School of Medicine.

TECHNICAL FIELD OF THE INVENTION

This invention relates to delivery of biologically active cargomolecules, such as polypeptides and nucleic acids, into the cytoplasmand nuclei of cells in vitro and in vivo. Intracellular delivery ofcargo molecules according to this invention is accomplished by the useof novel transport polypeptides which comprise HIV tat protein or one ormore portions thereof, and which are covalently attached to cargomolecules. The transport polypeptides in preferred embodiments of thisinvention are characterized by the presence of the tat basic region(amino acids 49-57), the absence of the tat cysteine-rich region (aminoacids 22-36) and the absence of the tat exon 2-encoded carboxy-terminaldomain (amino acids 73-86) of the naturally-occurring tat protein. Byvirtue of the absence of the cysteine-rich region, the preferredtransport polypeptides of this invention solve the potential problems ofspurious trans-activation and disulfide aggregation. The reduced size ofthe preferred transport polypeptides of this invention also minimizesinterference with the biological activity of the cargo molecule.

BACKGROUND OF THE INVENTION

Biological cells are generally impermeable to macromolecules, includingproteins and nucleic acids. Some small molecules enter living cells atvery low rates. The lack of means for delivering macromolecules intocells in vivo has been an obstacle to the therapeutic, prophylactic anddiagnostic use of a potentially large number of proteins and nucleicacids having intracellular sites of action. Accordingly, mosttherapeutic, prophylactic and diagnostic candidates produced to dateusing recombinant DNA technology are polypeptides that act in theextracellular environment or on the target cell surface.

Various methods have been developed for delivering macromolecules intocells in vitro. A list of such methods includes electroporation,membrane fusion with liposomes, high velocity bombardment withDNA-coated microprojectiles, incubation with calcium-phosphate-DNAprecipitate, DEAE-dextran mediated transfection, infection with modifiedviral nucleic acids, and direct micro-injection into single cells. Thesein vitro methods typically deliver the nucleic acid molecules into onlya fraction of the total cell population, and they tend to damage largenumbers of cells. Experimental delivery of macromolecules into cells invivo has been accomplished with scrape loading, calcium phosphateprecipitates and liposomes. However, these techniques have, to date,shown limited usefulness for in vivo cellular delivery. Moreover, evenwith cells in vitro, such methods are of extremely limited usefulnessfor delivery of proteins.

General methods for efficient delivery of biologically active proteinsinto intact cells, in vitro and in vivo, are needed. (L. A. Sternson,"Obstacles to Polypeptide Delivery", Ann. N.Y. Acad. Sci, 57, pp. 19-21(1987)). Chemical addition of a lipopeptide (P. Hoffmann et al.,"Stimulation of Human and Murine Adherent Cells by Bacterial Lipoproteinand Synthetic Lipopeptide Analogues", Immunobiol., 177, pp. 158-70(1988)) or a basic polymer such as polylysine or polyarginine (W.-C.Chen et al., "Conjugation of Poly-L-Lysine Albumin and HorseradishPeroxidase: A Novel Method of Enhancing the Cellular Uptake ofProteins", Proc. Natl. Acad. Sci. USA, 75, pp. 1872-76 (1978)) have notproved to be highly reliable or generally useful (see Example 4 infra,).Folic acid has been used as a transport moiety (C. P. Leamon and Low,Delivery of Macromolecules into Living Cells: A Method That ExploitsFolate Receptor Endocytosis", Proc. Natl. Acad. Sci USA, 88, pp. 5572-76(1991)). Evidence was presented for internalization of folateconjugates, but not for cytoplasmic delivery. Given the high levels ofcirculating folate in vivo, the usefulness of this system has not beenfully demonstrated. Pseudomonas exotoxin has also been used as atransport moiety (T. I. Prior et al., "Barnase Toxin: A New ChimericToxin Composed of Pseudomonas Exotoxin A and Barnase", Cell, 64, pp.1017-23 (1991)). The efficiency and general applicability of this systemfor the intracellular delivery of biologically active cargo molecules isnot clear from the published work, however.

Purified human immunodeficiency virus type-1 ("HIV") tat protein istaken up from the surrounding medium by human cells growing in culture(A. D. Frankel and C. O. Pabo, "Cellular Uptake of the Tat Protein fromHuman Immunodeficiency Virus", Cell, 55, pp. 1189-93 (1988)). Tatprotein trans-activates certain HIV genes and is essential for viralreplication. The full-length HIV-1 tat protein has 86 amino acidresidues. The HIV tat gene has two exons. Tat amino acids 1-72 areencoded by exon 1, and amino acids 73-86 are encoded by exon 2. Thefull-length tat protein is characterized by a basic region whichcontains two lysines and six arginines (amino acids 49-57) and acysteine-rich region which contains seven cysteine residues (amino acids22-37).

The basic region (i.e., amino acids 49-57) is thought to be importantfor nuclear localization. Ruben, S. et al., J. Virol. 63: 1-8 (1989);Hauber, J. et al., J. Virol. 63 1181-1187 (1989). The cysteine-richregion mediates the formation of metal-linked dimers in vitro (Frankel,A. D. et al, Science 240: 70-73 (1988); Frankel, A. D. et al., Proc.Natl. Acad. Sci USA 85: 6297-6300 (1988)) and is essential for itsactivity as a transactivator (Garcia, J. A. et al., EMBO J. 7: 3143(1988); Sadaie, M. R. et al., J. Virol. 63:1 (1989)). As in otherregulatory proteins, the N-terminal region may be involved in protectionagainst intracellular proteases (Bachmair, A. et al., Cell 56: 1019-1032(1989)).

At the present time, the need exists for generally applicable means forsafe, efficient delivery of biologically active molecule of interest orcargo molecules into the cytoplasm and nuclei of living cells.

SUMMARY OF THE INVENTION

The present invention relates to the use of HIV tat protein, or atat-derived polypeptide, to deliver a molecule of interest or cargomolecule into eukaryotic cells, particularly into the cell nucleus, invitro or in vivo. It further relates to conjugates that include an HIVtat protein and a molecule of interest, or a tat-derived polypeptide anda cargo molecule, which are useful in the method of the presentinvention for delivering biologically active molecules into thecytoplasm and nuclei of cells.

More particularly, this invention provides processes and products forthe efficient cytoplasmic and nuclear delivery of biologically activenon-tat proteins, nucleic acids and other molecules that are (1) notinherently capable of entering target cells or cell nuclei, or (2) notinherently capable of entering target cells at a useful rate.Intracellular delivery of cargo molecules according to this invention isaccomplished by the use of novel transport proteins which comprise oneor more portions of HIV tat protein and which are covalently attached tothe cargo molecules. According to various embodiments, this inventionrelates to novel transport polypeptides, methods for making thosetransport polypeptides, transport polypeptide-cargo conjugates,pharmaceutical, prophylactic and diagnostic compositions comprisingtransport polypeptide-cargo conjugates, and methods for delivery ofcargo into cells by means of tat-derived transport polypeptides.

The preferred transport polypeptides of this invention are characterizedby the presence of the tat basic region amino acid sequence (amino acids49-57 of naturally-occurring tat protein); the absence of the tatcysteine-rich region amino acid sequence (amino acids 22-36 ofnaturally-occurring tat protein) and the absence of the tat exon2-encoded carboxy-terminal domain (amino acids 73-86 ofnaturally-occurring tat protein). Preferred embodiments of suchtransport polypeptides are: tat37-72 (SEQ ID NO:2), tat37-58 (SEQ IDNO:3), tat38-58GGC (SEQ ID NO:4), tatCGG47-58 (SEQ ID NO:5) tat47-58GGC(SEQ ID NO:6), and tatΔcys (SEQ ID NO:7). It will be recognized by thoseof ordinary skill in the art that when the transport polypeptide isgenetically fused to the cargo moiety, an amino-terminal methionine mustbe added, but the spacer amino acids (e.g., CysGlyGly or GlyGlyCys) neednot be added.

By virtue of the absence of the cysteine-rich region present inconventional tat proteins, the preferred transport polypeptides of thisinvention solve the problem of disulfide aggregation, which can resultin loss of the cargo's biological activity, insolubility of thetransport polypeptide-cargo conjugate, or both. The reduced size of thepreferred transport polypeptides of this invention also advantageouslyminimizes interference with the biological activity of the cargo. Afurther advantage of the reduced transport polypeptide size is enhanceduptake efficiency in embodiments of this invention involving attachmentof multiple transport polypeptides per cargo molecule.

Transport polypeptides of this invention may be advantageously attachedto cargo molecules by chemical cross-linking or by genetic fusion. Aunique terminal cysteine residue is a preferred means of chemicalcross-linking. According to some preferred embodiments of thisinvention, the carboxy terminus of the transport moiety is geneticallyfused to the amino terminus of the cargo moiety. A particularlypreferred embodiment of the present invention is JB106, which consistsof an amino-terminal methionine followed by tat residues 47-58, followedby HPV-16 E2 residues 245-365.

According to one preferred embodiment of this invention, a biologicallyactive cargo is delivered into the cells of various organs and tissuesfollowing introduction of a transport polypeptide-cargo conjugate into alive human or animal. By virtue of the foregoing features, thisinvention opens the way for biological research and disease therapyinvolving proteins, nucleic acids and other molecules with cytoplasmicor nuclear sites of action.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the amino acid sequence of HIV-1 tat protein (SEQ IDNO:1).

FIG. 2 depicts a thin layer chromatogram showing chloramphenicol acetyltransferase activity, a measure of tat protein uptake, resulting fromincubating HL3T1 cells with tat protein for 24 hours at the indicatedconcentrations.

FIG. 3 depicts a thin layer chromatogram showing chloramphenicol acetyltransferase activity resulting from incubating HL3T1 cells with 5 μg oftat protein and a variety of lysosomotrophic agents.

FIG. 4 graphically depicts the cellular uptake and nuclear localizationof ¹²⁵ I-labeled tat protein.

FIG. 5 depicts an autoradiogram of an SDS polyacrylamide electrophoresisgel on which nuclear fractions from HL3T1 cells treated with tat proteinwere analyzed in the absence (-) or presence (+) of chloroquine.

FIG. 6A graphically depicts the effect of chloroquine concentration onuptake and transactivation by 2 μg tat protein added to the medium.

FIG. 6B graphically depicts the time course of uptake andtransactivation by 2 μg of tat protein and 100 μM chloroquine.

FIG. 6C graphically depicts the effect on uptake and transactivation byseveral concentrations of tat protein with 100 μM chloroquine.

FIG. 7 depicts a thin layer chromatogram used to analyze chloramphenicolacetyltransferase activity from H9 lymphocytes, U937 promonocytes andHeLa cells treated with tat protein in the absence or presence ofchloroquine.

FIG. 8 depicts a thin layer chromatogram used to analyze chloramphenicolacetyl transferase activity from HL3T1 cells in experiments illustratingthe extent of activation of the CAT reporter gene following various timeperiods of exposure to 1 μg tat protein+100 μM chloroquine.

FIG. 9 schematically depicts the murine sarcoma virus (MSV) retroviralvector used to establish the H938 reporter cell line from H9 cells. Thetranscription start sites from the SV40 promoter, the HIV and MSV LTRsare indicated by arrows, and the location and size of the fragmentsprotected in the RNase analysis are indicated by bars.

FIG. 10 depicts an autoradiogram from an RNase protection experiment inH938 cells, using an α-³² P UTP-labeled HIV-1 LTR probe corresponding toa 200 bp fragment from -120 to +80 of the viral LTR, prepared by invitro transcription.

FIG. 11 summarizes data on the enhancement of transactivation in H938cells upon addition of increasing amounts of the tat 38-58 peptide with1 μg of tat protein as assayed by CAT activity after a 24 hourincubation with peptide and tat protein.

FIG. 12 summarizes data on transactivation of a tPA reporter gene underthe control of the HIV-1 LTR in HeLa.318 cells upon addition ofexogenous tat protein or a tatE2C fusion protein.

FIG. 13 summarizes the results of cellular uptake experiments withtransport polypeptide-Pseudomonas exotoxin ribosylation domainconjugates (shaded bars, unconjugated; diagonally-hatched bars,conjugated).

FIG. 14 summarizes the results of cellular uptake experiments withtransport polypeptide-ribonuclease conjugates (closed squares,ribonuclease-SMCC without transport moiety; closed circles,tat37-72-ribonuclease; closed triangles tat38-58GGC-ribonuclease; closeddiamonds, tatCGG38-58-ribonuclease; open squares,tatCGG47-58-ribonuclease).

FIG. 15 schematically depicts the construction of plasmid pAHE2.

FIG. 16 schematically depicts the construction of plasmid pET8c123.

FIG. 17 schematically depicts the construction of plasmid pET8c123CCSS.

FIG. 18 summarizes the results of cellular uptake experiments withtransport polypeptide-E2 repressor conjugates (open diamonds, E2.123cross-linked to tat37-72, without chloroquine; closed diamonds, E2.123cross-linked to tat37-72, with chloroquine; open circles, E2.123CCSScross-linked to tat37-72, without chloroquine; closed circles,E2.123CCSS cross-linked to tat37-72, with chloroquine).

FIG. 19 schematically depicts the construction of plasmid pTATΔcys.

FIG. 20 schematically depicts the construction of plasmid pFTE501.

FIG. 21 schematically depicts the construction of plasmid pTATΔcys-249.

FIG. 22 schematically depicts the construction of plasmid pJB106.

FIG. 23 depicts the complete amino acid sequence of protein JB106. (SEQID No:38)

FIG. 24 summarizes the results of E2 repression assays involving JB106(squares), TxHE2CCSS (diamonds) and HE2.123 (circles). The assays werecarried out in COS7 cells, without chloroquine, as described in Example14.

DETAILED DESCRIPTION OF THE INVENTION

In order that the invention herein described may be more fullyunderstood, the following detailed description is set forth.

In the description, the following terms are employed:

Amino acid--A monomeric unit of a peptide, polypeptide or protein. Thetwenty protein amino acids (L-isomers) are: alanine ("Ala" or "A"),arginine ("Arg" or "R"), asparagine ("Asn" or "N"), aspartic acid ("Asp"or "D"), cysteine ("Cys" or "C"), glutamine ("Gln" or "Q"), glutamicacid ("Glu" or "E"), glycine ("Gly" or "G"), histidine ("His" or "H"),isoleucine ("Ile" or "I"), leucine ("Leu" or "L"), lysine ("Lys" or"K"), methionine ("Met" or "M"), phenylalanine ("Phe" or "F"), proline("Pro" or "P"), serine ("Ser" or "S"), threonine ("Thr" or "T"),tryptophan ("Trp" or "W"), tyrosine ("Tyr" or "Y") and valine ("Val" or"V"). The term amino acid, as used herein, also includes analogs of theprotein amino acids, and D-isomers of the protein amino acids and theiranalogs.

Cargo--A molecule that is not a tat protein or a fragment thereof, andthat is either (1) not inherently capable of entering target cells, or(2) not inherently capable of entering target cells at a useful rate.("Cargo", as used in this application, refers either to a molecule, perse, i.e., before conjugation, or to the cargo moiety of a transportpolypeptide-cargo conjugate.) Examples of "cargo" include, but are notlimited to, small molecules and macromolecules, such as polypeptides,nucleic acids and polysaccharides.

Chemical cross-linking--Covalent bonding of two or more pre-formedmolecules.

Cargo conjugate--A molecule comprising at least one transportpolypeptide moiety and at least one cargo moiety, formed either throughgenetic fusion or chemical cross-linking of a transport polypeptide anda cargo molecule.

Genetic fusion--Co-linear, covalent linkage of two or more proteins viatheir polypeptide backbones, through genetic expression of a DNAmolecule encoding those proteins.

Macromolecule--A molecule, such as a peptide, polypeptide, protein ornucleic acid.

Molecule of interest--See definition of cargo, above.

Polypeptide--Any polymer consisting essentially of any of the 20 proteinamino acids (above), regardless of its size. Although "protein" is oftenused in reference to relatively large polypeptides, and "peptide" isoften used in reference to small polypeptides, usage of these terms inthe art overlaps and varies. The term "polypeptide" as used hereinrefers to peptides, polypeptides and proteins, unless otherwise noted.

Reporter gene--A gene the expression of which depends on the occurrenceof a cellular event of interest, and the expression of which can beconveniently observed in a genetically transformed host cell.

Reporter plasmid--A plasmid vector comprising one or more reportergenes.

Small molecule--A molecule other than a macromolecule.

Spacer amino acid--An amino acid (preferably having a small side chain)included between a transport moiety and an amino acid residue used forchemical cross-linking (e.g., to provide molecular flexibility and avoidsteric hindrance).

Target cell--A cell into which a cargo is delivered by a transportpolypeptide. A "target cell" may be any cell, including human cells,either in vivo or in vitro.

Transport moiety or transport polypeptide--A polypeptide capable ofdelivering a covalently attached cargo into a target cell, e.g., tatprotein or a tat-derived polypeptide.

The present invention is based on the unexpected finding that when tatprotein from immunodeficiency virus (e.g., HIV-1, HIV-2, SIV) is presentextracellularly, it is readily taken up into cells and subsequently intothe cell nucleus. This is evidenced by the fact that when cultured cellswith an integrated HIV-1 promoter are treated with tat in the medium,they exhibit high levels of transactivation of the HIV-1 promoter. Inlight of the fact that proteins and peptides are typically poorly takenup (Sternson, L. A., Ann. N.Y. Acad. Sci. 57: 19-21 (1987)), the findingthat tat is readily taken up into cells is surprising.

As a result of this finding, it is now possible to use tat protein todeliver molecules (e.g., proteins, peptides, nucleic acids) into cellsand, specifically, into the cell nucleus. The present invention relatesto a method of delivering a molecule of interest into cells and,particularly, of targeting a molecule to the cell nucleus, as well as aconjugate useful in the method. Any molecule can be delivered intocells, especially into the cell nucleus, using the method of the subjectinvention. For example, in one embodiment of the present method, themolecule to be delivered into cells is a protein, a peptide or anoligonucleotide. The present invention is particularly useful fordelivery of proteins or peptides, such as regulatory factors, enzymes,antibodies, drugs or toxins, as well as DNA or RNA, into the cellnucleus.

A stabilizing agent, which serves to increase tat stability and uptake,can be brought into contact with cells, in conjunction with the moleculeof interest and tat protein. For example, metal ions which bind to tatprotein and increase its stability and uptake, can be used for thispurpose.

In a further embodiment of this invention, a lysosomotrophic agent isprovided extracellularly in conjunction with tat protein and a moleculeof interest, in order to enhance uptake by cells. The lysosomotrophicagent can be used alone or in conjunction with a stabilizer. Forexample, lysosomotrophic agents such as chloroquine, monensin,amantadine and methylamine, which have been shown to increase uptake oftat in some cells by a few hundred fold, can be used for this purpose.

In another embodiment, a basic peptide, such as tat 38-58 or protamine,is provided extracellularly with tat and a molecule of interest toenhance uptake of Tat. Such basic peptides can also be used alone, incombination or with stabilizing agents or lysosomotrophic agents.

In one embodiment of the present invention, a molecule of interest-tatprotein conjugate, which includes a molecule of interest (i.e., amolecule to be introduced into cells) attached to HIV tat protein, isbrought into contact with cells into which introduction of the moleculeof interest is desired, under conditions appropriate for its entry intocells. As a result, the conjugate enters into cells, passing into thenucleus.

The present invention may be used to deliver a molecule of interesteither in vitro or in vivo. For example, delivery can be carried out invitro by adding a molecule of interest-tat conjugate to cultured cells,by producing cells that synthesize tat or tat conjugate or by combininga sample (e.g., blood, bone marrow) obtained from an individual with theconjugate, under appropriate conditions. Thus, the target cells may bein vitro cells, i.e., cultured animal cells, human cells ormicro-organisms. Delivery can be carried out in vivo by administeringthe molecule of interest and tat protein to an individual in whom it isto be used for diagnostic, preventative or therapeutic purposes. Thetarget cells may be in vivo cells, i.e., cells composing the organs ortissues of living animals or humans, or microorganisms found in livinganimals or humans.

This invention is generally applicable for therapeutic, prophylactic ordiagnostic intracellular delivery of small molecules and macromolecules,such as proteins, nucleic acids and polysaccharides, that are notinherently capable of entering target cells at a useful rate. It shouldbe appreciated, however, that alternate embodiments of this inventionare not limited to clinical applications. This invention may beadvantageously applied in medical and biological research. In researchapplications of this invention, the cargo may be a drug or a reportermolecule. Transport polypeptides of this invention may be used asresearch laboratory reagents, either alone or as part of a transportpolypeptide conjugation kit.

Wide latitude exists in the selection of drugs and reporter moleculesfor use in the practice of this invention. Factors to be considered inselecting reporter molecules include, but are not limited to, the typeof experimental information sought, non-toxicity, convenience ofdetection, quantifiability of detection, and availability. Many suchreporter molecules are known to those skilled in the art.

As will be appreciated from the examples presented below, we have usedenzymes for which calorimetric assays exist, as model cargo todemonstrate the operability and useful features of the transportpolypeptides of this invention. These enzyme cargos provide forsensitive, convenient, visual detection of cellular uptake. Furthermore,since visual readout occurs only if the enzymatic activity of the cargois preserved, these enzymes provide a sensitive and reliable test forpreservation of biological activity of the cargo moiety in transportpolypeptide-cargo conjugates according to this invention. A preferredembodiment of this invention comprises horseradish peroxidase ("HRP") asthe cargo moiety of the transport polypeptide-cargo conjugate. Aparticularly preferred model cargo moiety for practice of this inventionis β-galactosidase.

Model cargo proteins may also be selected according to their site ofaction within the cell. As described in Examples 6 and 7, below, we haveused the ADP ribosylation domain from Pseudomonas exotoxin ("PE") andpancreatic ribonuclease to confirm cytoplasmic delivery of a properlyfolded cargo proteins by transport polypeptides according to thisinvention.

Full-length Pseudomonas exotoxin is itself capable of entering cells,where it inactivates ribosomes by means of an ADP ribosylation reaction,thus killing the cells. A portion of the Pseudomonas exotoxin proteinknown as the ADP ribosylation domain is incapable of entering cells, butit retains the ability to inactivate ribosomes if brought into contactwith them. Thus, cell death induced by transport polypeptide-PE ADPribosylation domain conjugates is a test for cytoplasmic delivery of thecargo by the transport polypeptide.

We have also used ribonuclease to confirm cytoplasmic delivery of aproperly folded cargo protein by transport polypeptides of thisinvention. Protein synthesis, an RNA-dependent process, is highlysensitive to ribonuclease, which digests RNA. Ribonuclease is, byitself, incapable of entering cells, however. Thus, inhibition ofprotein synthesis by a transport polypeptide-ribonuclease conjugate is atest for intracellular delivery of biologically active ribonuclease.

Of course, delivery of a given cargo molecule to the cytoplasm may befollowed by further delivery of the same cargo molecule to the nucleus.Nuclear delivery necessarily involves traversing some portion of thecytoplasm.

Papillomavirus E2 repressor proteins are examples of macromoleculardrugs that may be delivered into the nuclei of target cells by thetransport polypeptides of this invention. Papillomavirus E2 protein,which normally exists as a homodimer, regulates both transcription andreplication of the papillomavirus genome. The carboxy-terminal domain ofthe E2 protein contains DNA binding and dimerization activities.Transient expression of DNA sequences encoding various E2 analogs or E2carboxy-terminal fragments in transfected mammalian cells inhibitstrans-activation by the full-length E2 protein (J. Barsoum et al.,"Mechanism of Action of the Papillomavirus E2 Repressor: Repression inthe Absence of DNA Binding", J. Virol., 66, pp. 3941-3945 (1992)). E2repressors added to the growth medium of cultured mammalian cells do notenter the cells, and thus do not inhibit E2 trans-activation in thosecells. However, conjugation of the transport polypeptides of thisinvention to E2 repressors results in translocation of the E2 repressorsfrom the growth medium into the cultured cells, where they displaybiological activity, repressing E2-dependent expression of a reportergene.

The rate at which single-stranded and double-stranded nucleic acidsenter cells, in vitro and in vivo, may be advantageously enhanced, usingthe transport polypeptides of this invention. As shown in Example 11(below), methods for chemical cross-linking of polypeptides to nucleicacids are well known in the art. In a preferred embodiment of thisinvention, the cargo is a single-stranded antisense nucleic acid.Antisense nucleic acids are useful for inhibiting cellular expression ofsequences to which they are complementary. In another embodiment of thisinvention, the cargo is a double-stranded nucleic acid comprising abinding site recognized by a nucleic acid-binding protein. An example ofsuch a nucleic acid-binding protein is a viral trans-activator.

It will be appreciated that the entire 86 amino acids which make up thetat protein may not be required for the uptake activity of tat. Forexample, a protein fragment or a peptide which has fewer than the 86amino acids, but which exhibits uptake into cells and uptake into thecell nucleus, can be used (a functionally effective fragment or portionof tat). As is shown in the Examples below, tat protein containingresidues 1-72 is sufficient for uptake activity and tat residues 1-67are shown to mediate the entry of a heterologous protein into cells. Inaddition, a synthetic peptide containing tat residues 1-58 has now beenshown to have uptake activity. A tat peptide comprising the region thatmediates entry and uptake into cells can be further defined using knowntechniques (see, e.g., Frankel, A. D., et al., Proc. Natl. Acad. Sci,USA 86: 7397-7401 (1989)).

The tat peptide can be a single (i.e., continuous) amino acid sequencepresent in tat protein or it can be two or more amino acid sequenceswhich are present in tat protein, but in the naturally-occurring proteinare separated by other amino acid sequences. As used herein, tat proteinincludes a naturally-occurring amino acid sequence which is the same asthat of naturally-occurring tat protein, its functional equivalent orfunctionally equivalent fragments thereof (peptides). Such functionalequivalents or functionally equivalent fragments possess uptake activityinto the cell and into the cell nucleus that is substantially similar tothat of naturally-occurring tat protein. Tat protein can be obtainedfrom naturally-occurring sources or can be produced using geneticengineering techniques or chemical synthesis.

The amino acid sequence of naturally-occurring HIV tat protein can bemodified, by addition, deletion and/or substitution of at least oneamino acid present in the naturally-occurring tat protein, to producemodified tat protein (also referred to herein as tat protein). Modifiedtat protein or tat peptide analogs with increased stability can thus beproduced using known techniques. Therefore, tat proteins or peptides mayhave amino acid sequences which are substantially similar, although notidentical, to that of naturally-occurring tat protein or portionsthereof. In addition, cholesterol or other lipid derivatives can beadded to tat protein to produce a modified tat having increased membranesolubility.

Variants of tat protein can be designed to modulate the intracellularlocation of tat and the molecule of interest following uptake into thecell or when expressed in the cell. When added exogenously, suchvariants are designed such that the ability of tat to enter cells isretained (i.e., the uptake of the variant tat protein or peptide intothe cell is substantially similar to that of naturally-occurring HIVtat). For example, alteration of the basic region thought to beimportant for nuclear localization (see e.g., Dang, C. V. and Lee, W. M.F., J. Biol. Chem. 264: 18019-18023 (1989); Hauber, J. et al., J.Virol.63: 1181-1187 (1989); Ruben, S. A. et al., J. Virol. 63: 1-8 (1989)) canresult in a cytoplasmic location or partially cytoplasmic location oftat, and therefore, of the molecule of interest. Alternatively, asequence for binding a cytoplasmic component can be introduced into tatin order to retain tat and the molecule of interest in the cytoplasm orto confer regulation upon nuclear uptake of tat and the molecule ofinterest.

Naturally-occurring HIV-1 tat protein (FIG. 1) has a region (amino acids22-37) wherein 7 out of 16 amino acids are cysteine. Those cysteineresidues are capable of forming disulfide bonds with each other, withcysteine residues in the cysteine-rich region of other tat proteinmolecules and with cysteine residues in a cargo protein or the cargomoiety of a conjugate. Such disulfide bond formation can cause loss ofthe cargo's biological activity. Furthermore, even if there is nopotential for disulfide bonding to the cargo moiety (for example, whenthe cargo protein has no cysteine residues), disulfide bond formationbetween transport polypeptides leads to aggregation and insolubility ofthe transport polypeptide, the transport polypeptide-cargo conjugate, orboth. The tat cysteine-rich region is potentially a source of seriousproblems in the use of naturally-occurring tat protein for cellulardelivery of cargo molecules.

The cysteine-rich region is required for dimerization of tat in vitro,and is required for trans-activation of HIV DNA sequences. Therefore,removal of the tat cysteine-rich region has the additional advantage ofeliminating the natural activity of tat, i.e., induction of HIVtranscription and replication. However, the art does not teach whetherthe cysteine-rich region of the tat protein is required for cellularuptake.

The present invention includes embodiments wherein any problemsassociated with the tat cysteine-rich region are solved, because thatregion is not present in the transport polypeptides described herein. Inthose embodiments, cellular uptake of the transport polypeptide ortransport polypeptide-cargo molecule conjugate still occurs. In onegroup of preferred embodiments of this invention, the sequence of aminoacids preceding the cysteine-rich region is fused directly to thesequence of amino acids following the cysteine-rich region. Suchtransport polypeptides are called tatΔcys, and have the general formula(tat1-21)-(tat38-n), where n is the number of the carboxy-terminalresidue, i.e., 49-86. Preferably, n is 58-72. As will be appreciatedfrom the examples below, the amino acid sequence preceding thecysteine-rich region of the tat protein is not required for cellularuptake. A preferred transport polypeptide (or transport moiety) consistsof amino acids 37-72 of tat protein, and is called tat37-72 (SEQ IDNO:2). Retention of tat residue 37, a cysteine, at the amino terminus ofthe transport polypeptide is preferred, because it is useful forchemical cross-linking.

The advantages of the tatΔcys polypeptides, tat37-72 and otherembodiments of this invention include the following:

a) The natural activity of tat protein, i.e., induction of HIVtranscription, is eliminated;

b) Dimers, and higher multimers of the transport polypeptide areavoided;

c) The level of expression of tatΔcys genetic fusions in E.coli may beimproved;

d) Some transport polypeptide conjugates display increased solubilityand superior ease of handling; and

e) Some fusion proteins display increased activity by the cargo moiety,as compared with fusions containing the cysteine-rich region.

The pharmaceutical compositions of this invention may be fortherapeutic, prophylactic or diagnostic applications, and may be in avariety of forms. These include, for example, solid, semi-solid, andliquid dosage forms, such as tablets, pills, powders, liquid solutionsor suspensions, aerosols, liposomes, suppositories, injectable andinfusible solutions and sustained release forms. The preferred formdepends on the intended mode of administration and the therapeutic,prophylactic or diagnostic application. According to this invention, aselected molecule of interest-tat protein conjugate or a transportpolypeptide-cargo molecule conjugate may be administered by conventionalroutes of administration, such as parenteral, subcutaneous, intravenous,intramuscular, intralesional, intrasternal, intracranial or aerosolroutes. Topical routes of administration may also be used, withapplication of the compositions locally to a particular part of the body(e.g., skin, lower intestinal tract, vagina, rectum) where appropriate.In the case of a papillomavirus infection, for example, topicaladministration would be indicated. The compositions also preferablyinclude conventional pharmaceutically acceptable carriers and adjuvantsthat are known to those of skill in the art.

A selected molecule of interest in combination with tat protein or amolecule of interest-tat protein conjugate can also be used in making avaccine. For example, the molecule of interest can be an antigen fromthe bacteria or virus or other infectious agent that the vaccine is toimmunize against (e.g., gp120 of HIV). Providing the antigen into thecell cytoplasm allows the cell to process the molecule and express it onthe cell surface. Expression of the antigen on the cell surface willraise a killer T-lymphocyte response, thereby inducing immunity.

Generally, the pharmaceutical compositions of the present invention maybe formulated and administered using methods and compositions similar tothose used for pharmaceutically important polypeptides such as, forexample, alpha interferon. It will be understood that conventional doseswill vary depending upon the particular cargo involved, as well as thepatient's health, weight, age, sex, the condition or disease and thedesired mode of administration. The pharmaceutical compositions of thisinvention include pharmacologically appropriate carriers, adjuvants andvehicles. In general, these carriers include aqueous oralcoholic/aqueous solutions, emulsions or suspensions, including salineand buffered media. Parenteral vehicles can include sodium chloridesolution, Ringer's dextrose, dextrose and sodium chloride, lactatedRinger's or fixed oils. In addition, intravenous vehicles can includefluid and nutrient replenishers, and electrolyte replenishers, such asthose based on Ringer's dextrose. Preservatives and other additives canalso be present, such as, for example, antimicrobials, antioxidants,chelating agents, and inert gases. See, generally, Remington'sPharmaceutical Sciences, 16th Ed., Mack, ed. 1980.

The processes and compositions of this invention may be applied to anyorganism, including humans. The processes and compositions of thisinvention may also be applied to animals and humans in utero.

For many pharmaceutical applications of this invention, it is necessaryfor the cargo molecule to be translocated from body fluids into cells oftissues in the body, rather than from a growth medium into culturedcells. Therefore, in addition to examples below involving culturedcells, we have provided examples demonstrating delivery of model cargoproteins into cells of various mammalian organs and tissues, followingintravenous injection of transport polypeptide-cargo protein conjugatesinto live animals. These cargo proteins display biological activityfollowing delivery into the cells in vivo.

As demonstrated in the examples that follow, using the amino acid andDNA sequence information provided herein, the transport polypeptides ofthis invention may be chemically synthesized or produced by recombinantDNA methods. Methods for chemical synthesis or recombinant DNAproduction of polypeptides having a known amino acid sequence are wellknown. Automated equipment for polypeptide or DNA synthesis iscommercially available. Host cells, cloning vectors, DNA expressioncontrol sequences and oligonucleotide linkers are also commerciallyavailable.

Using well-known techniques, one of skill in the art can readily makeminor additions, deletions or substitutions in the preferred transportpolypeptide amino acid sequences set forth herein. It should beunderstood, however, that such variations are within the scope of thisinvention.

Furthermore, tat proteins from other viruses, such as HIV-2 (M. Guyaderet al., "Genome Organization and Transactivation of the HumanImmunodeficiency Virus Type 2", Nature, 326, pp. 662-669 (1987)), equineinfectious anemia virus (R. Carroll et al., "Identification ofLentivirus Tat Functional Domains Through Generation of EquineInfectious Anemia Virus/Human Immunodeficiency Virus Type 1 tat GeneChimeras", J. Virol., 65, pp. 3460-67 (1991)), and simianimmunodeficiency virus (L. Chakrabarti et al., "Sequence of SimianImmunodeficiency Virus from Macaque and Its Relationship to Other Humanand Simian Retroviruses", Nature, 328, pp. 543-47 (1987); S. K. Arya etal., "New Human and Simian HIV-Related Retroviruses Possess FunctionalTransactivator (tat) Gene", Nature, 328, pp. 548-550 (1987)) are known.It should be understood that polypeptides derived from those tatproteins fall within the scope of the present invention, including thosecharacterized by the presence of the tat basic region and the absence ofthe tat cysteine-rich region.

The Molecule of Interest-Tat Protein Conjugate

A molecule of interest, which will generally be a protein or peptide, anucleotide sequence, or other chemical which has diagnostic,prophylactic or therapeutic application (referred to herein as a drug)is combined, as described below, with HIV tat protein to produce amolecule of interest-tat protein conjugate. The resulting conjugate isbrought into contact with the extracellular surface of cells.

In one embodiment of the present invention, the molecule of interest isa protein, such as an enzyme, antibody, toxin, or regulatory factor(e.g., transcription factor) whose delivery into cells, and particularlyinto the cell nucleus is desired. For example, some viral oncogenesinappropriately turn on expression of cellular genes by binding to theirpromoters. By providing a competing binding protein in the cell nucleus,viral oncogene-activity can be inhibited.

In a further embodiment, the molecule of interest is a nucleotidesequence to be used as a diagnostic tool (or probe), or as a therapeuticagent, such as an oligonucleotide sequence which is complementary to atarget cellular gene or gene region and capable of inhibiting activityof the cellular gene or gene region by hybridizing with it. In yetanother embodiment, the molecule of interest is a drug, such as apeptide analog or small molecule enzyme inhibitor, whose introductionspecifically and reliably into the cell nucleus is desired.

The molecule of interest can be obtained or produced using knowntechniques, such as chemical synthesis, genetic engineering methods andisolation from sources in which it occurs naturally. The molecule ofinterest can be combined with or attached to the tat protein to form themolecule of interest-tat protein conjugate which is a subject of thepresent invention.

The attachment of the molecule of interest to tat to produce a moleculeof interest-tat protein conjugate may be effected by any means whichproduces a link between the two constituents which is sufficientlystable to withstand the conditions used and which does not alter thefunction of either constituent. Preferably, the link between them iscovalent. For example, recombinant techniques can be used to covalentlyattach tat protein to molecules, such as by joining the gene coding forthe molecule of interest with the gene coding for tat and introducingthe resulting gene construct into a cell capable of expressing theconjugate. Alternatively, the two separate nucleotide sequences can beexpressed in a cell or can be synthesized chemically and subsequentlyjoined, using known techniques. Alternatively, the protein ofinterest-tat molecule can be synthesized chemically as a single aminoacid sequence (i.e., one in which both constituents are present) and,thus, joining is not needed.

Numerous chemical cross-linking methods are known and potentiallyapplicable for conjugating the transport polypeptides of this inventionto cargo macromolecules. Many known chemical cross-linking methods arenon-specific, i.e., they do not direct the point of coupling to anyparticular site on the transport polypeptide or cargo macromolecule. Asa result, use of non-specific cross-linking agents may attack functionalsites or sterically block active sites, rendering the conjugatedproteins biologically inactive.

A preferred approach to increasing coupling specificity in the practiceof this invention is direct chemical coupling to a functional groupfound only once or a few times in one or both of the polypeptides to becross-linked. For example, in many proteins, cysteine, which is the onlyprotein amino acid containing a thiol group, occurs only a few times.Also, for example, if a polypeptide contains no lysine residues, across-linking reagent specific for primary amines will be selective forthe amino terminus of that polypeptide. Successful utilization of thisapproach to increase coupling specificity requires that the polypeptidehave the suitably rare and reactive residues in areas of the moleculethat may be altered without loss of the molecule's biological activity.

As demonstrated in the examples below, cysteine residues may be replacedwhen they occur in parts of a polypeptide sequence where theirparticipation in a cross-linking reaction would likely interfere withbiological activity. When a cysteine residue is replaced, it istypically desirable to minimize resulting changes in polypeptidefolding. Changes in polypeptide folding are minimized when thereplacement is chemically and sterically similar to cysteine. For thesereasons, serine is preferred as a replacement for cysteine. Asdemonstrated in the examples below, a cysteine residue may be introducedinto a polypeptide's amino acid sequence for cross-linking purposes.When a cysteine residue is introduced, introduction at or near the aminoor carboxy terminus is preferred. Conventional methods are available forsuch amino acid sequence modifications, whether the polypeptide ofinterest is produced by chemical synthesis or expression of recombinantDNA.

Coupling of the two constituents can be accomplished via a coupling orconjugating agent. There are several intermolecular cross-linkingreagents which can be utilized (see, for example, Means, G. E. andFeeney, R. E., Chemical Modification of Proteins, Holden-Day, 1974, pp.39-43). Among these reagents are, for example, J-succinimidyl3-(2-pyridyldithio) propionate (SPDP) or N, N'-(1,3-phenylene)bismaleimide (both of which are highly specific for sulfhydryl groupsand form irreversible linkages); N, N'-ethylene-bis-(iodoacetamide) orother such reagent having 6 to 11 carbon methylene bridges (whichrelatively specific for sulfhydryl groups); and1,5-difluoro-2,4-dinitrobenzene (which forms irreversible linkages withamino and tyrosine groups). Other cross-linking reagents useful for thispurpose include: p,p'-difluoro-m,m'-dinitrodiphenylsulfone (which formsirreversible cross-linkages with amino and phenolic groups); dimethyladipimidate (which is specific for amino groups);phenol-1,4-disulfonylchloride (which reacts principally with aminogroups); hexamethylenediisocyanate or diisothiocyanate, orazophenyl-p-diisocyanate (which reacts principally with amino groups);glutaraldehyde (which reacts with several different side chains) anddisdiazobenzidine (which reacts primarily with tyrosine and histidine).

Cross-linking reagents may be homobifunctional, i.e., having twofunctional groups that undergo the same reaction. A preferredhomobifunctional cross-linking reagent is bismaleimidohexane ("BMH").BMH contains two maleimide functional groups, which react specificallywith sulfhydryl-containing compounds under mild conditions (pH 6.5-7.7).The two maleimide groups are connected by a hydrocarbon chain.Therefore, BMH is useful for irreversible cross-linking of polypeptidesthat contain cysteine residues.

Cross-linking reagents may also be heterobifunctional.Heterobifunctional cross-linking agents have two different functionalgroups, for example an amine-reactive group and a thiol-reactive group,that will cross-link two proteins having free amines and thiols,respectively. Examples of heterobifunctional cross-linking agents aresuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate ("SMCC"),m-maleimidobenzoyl-N-hydroxysuccinimide ester ("MBS"), and succinimide4-(p-maleimidophenyl)butyrate ("SMPB"), an extended chain analog of MBS.The succinimidyl group of these cross-linkers reacts with a primaryamine, and the thiol-reactive maleimide forms a covalent bond with thethiol of a cysteine residue.

Cross-linking reagents often have low solubility in water. A hydrophilicmoiety, such as a sulfonate group, may be added to the cross-linkingreagent to improve its water solubility. Sulfo-MBS and sulfo-SMCC areexamples of cross-linking reagents modified for water solubility.

Many cross-linking reagents yield a conjugate that is essentiallynon-cleavable under cellular conditions. However, some cross-linkingreagents contain a covalent bond, such as a disulfide, that is cleavableunder cellular conditions. For example,dithiobis(succinimidylpropionate) ("DSP"), Traut's reagent andN-succinimidyl 3-(2-pyridyldithio) propionate ("SPDP") are well-knowncleavable cross-linkers. The use of a cleavable cross-linking reagentpermits the cargo moiety to separate from the transport polypeptideafter delivery into the target cell. Direct disulfide linkage may alsobe useful.

Some new cross-linking reagents such asn-γ-maleimidobutyryloxy-succinimide ester ("GMBS") and sulfo-GMBS, havereduced immunogenicity. In some embodiments of the present invention,such reduced immunogenicity may be advantageous.

Numerous cross-linking reagents, including the ones discussed above, arecommercially available. Detailed instructions for their use are readilyavailable from the commercial suppliers. A general reference on proteincross-linking and conjugate preparation is: S. S. Wong, Chemistry ofProtein Conjugation and Cross-Linking, CRC Press (1991).

Chemical cross-linking may include the use of spacer arms. Spacer armsprovide intramolecular flexibility or adjust intramolecular distancesbetween conjugated moieties and thereby may help preserve biologicalactivity. A spacer arm may be in the form of a polypeptide moietycomprising spacer amino acids. Alternatively, a spacer arm may be partof the cross-linking reagent, such as in "long-chain SPDP" (Pierce Chem.Co., Rockford, Ill., cat. No. 21651 H).

Delivery of a Molecule of Interest Using the Present Method

The present method can be used to deliver a molecule of interest intocells, particularly into the cell nucleus, in vitro or in vivo. In invitro applications in which the molecule is to be delivered into cellsin culture, the molecule of interest in combination with tat protein orthe molecule of interest-tat protein conjugate is simply added to theculture medium. This is useful, for example, as a means of deliveringinto the nucleus substances whose effect on cell function is to beassessed. For example, the activity of purified transcription factorscan be measured, or the in vitro assay can be used to provide animportant test of a molecule's activity, prior to its use in in vivotreatment.

Alternatively, the molecule of interest in combination with tat proteinor the molecule of interest-tat protein conjugate can be used forprophylactic or therapeutic purposes (for the treatment, prophylaxis ordiagnosis of a disease or condition). For example, a selected moleculeof interest in combination with tat protein or the molecule ofinterest-tat protein conjugate can be combined with a sample obtainedfrom an individual (e.g., blood, bone marrow) in order to introduce themolecule of interest into cells present in the sample and, aftertreatment in this manner, the sample returned to the individual. Aseries of treatments carried out in this manner can be used to preventor inhibit the effects of an infectious agent. For example, blood can beremoved from an individual infected with HIV or other viruses, or froman individual with a genetic defect. The blood can then be combined witha molecule of interest in combination with tat protein or a molecule ofinterest-tat protein conjugate in which the molecule of interest is adrug capable of inactivating the virus or an oligonucleotide sequencecapable of hybridizing to a selected virus sequence and inactivating itor a protein that supplements a missing or defective protein, underconditions appropriate for entry in cells of the conjugate andmaintenance of the sample in such a condition that it can be returned tothe individual. After treatment, the blood is returned to theindividual.

Alternatively, the molecule of interest in combination with tat proteinor a molecule of interest-tat protein conjugate can be delivered invivo. For example, cells that synthesize tat or tat conjugate can beproduced and implanted into an individual so that tat or tat conjugateis constantly present. In another embodiment, the conjugate can be usedmuch like a conventional therapeutic agent and can be a component of apharmaceutical composition which includes other components useful, forexample, for delivery, stability or activity of the conjugate. In thisembodiment, a selected molecule of interest in combination with tatprotein or a molecule of interest-tat protein conjugate, such as aselected oligonucleotide sequence-tat protein conjugate, can beadministered in sufficient quantity to result in entry into cells,particularly cell nuclei, and inhibition (reduction or elimination) ofthe causative agent (e.g., virus or bacterium) or provision of a missingor defective protein.

Demonstration of Uptake of Tat into the Cell Nucleus

An unexpected result was seen when tat was simply added to the culturemedium of HL3T1 cells (HeLa cells containing the integrated LTR-CATplasmid). Expression of CAT from the integrated HIV-1 promoter increasedand was proportional to the tat concentration, indicating that tat wastaken up and transactivated the HIV-1 promoter. This result wassurprising because proteins and peptides are generally believed to bepoorly taken up by cells. Sternson, L. A., Ann. N.Y. Acad. Sci., 5719-21(1987).

To measure cellular uptake directly, HL3T1 cells were treated with ¹²⁵I-labeled tat in the presence or absence of chloroquine, and the amountof radioactive tat present in various cellular fractions was determined.This work is described in greater detail in Example III.

FIG. 2 shows results of assessment of the expression of CAT by HL3T1cells incubated with tat protein for 24 hours, at the indicatedconcentrations. Expression of CAT from the integrated HIV-1 promoterincreased and was proportional to the tat concentration. CAT activitydid not increase further after 24 hours. Additional small increases inactivity (2- to 3-fold) were observed upon addition of 10 mM zinc or 1mM cadmium, suggesting that metals might stabilize tat either duringuptake or once inside the cell.

To explore the uptake process further, various lysosomotrophic agentswere added to the culture medium. Lysosomotrophic agents are thought toinhibit receptor-mediated endocytosis. Mellman, I. et al., Ann. Rev.Biochem. 55: 663-700 (1986).

FIG. 3 shows the effect that a variety of lysosomotrophic agents have onuptake and subsequent transactivation by tat placed in tissue culturemedium. HL3T1 cells were incubated with 5 μg of tat (100 nM) and eachagent for 24 hours, the medium was replaced, and CAT activity wasdetermined after 60 hours. Activity from untreated cells, cellsincubated with tat alone and cells incubated with chloroquine alone arealso shown. The level of uptake and subsequent transactivation in HL3T1cells by 5 μg of tat with chloroquine present was about 7000-foldcompared with untreated cells, whereas chloroquine gave little increasein promoter activity in the absence of tat. Monensin, amantadine andmethylamine also significantly increased transactivation, whereasammonium chloride only slightly increased activity. No lysosomotrophicagent tested significantly activated the promoter in the absence of tat.The parameters of chloroquine-stimulated tat activity are explained inmore detail in Example IV.

FIG. 4 shows that within 6 hours after treating HL3T1 cells with tat andchloroquine, a significant amount of radioactive tat (about 3% of thetotal) had been taken up by the cells. Most of this tat (<80%) waslocalized in the nuclear fraction. Trypsin-sensitive counts,representing tat protein bound to the cell surface, remained relativelyconstant and by 12 hours were less than 20% of the counts found in thenucleus.

FIG. 5 shows nuclear extracts run on an SDS gel. A radioactive bandcomigrating with intact tat is readily apparent. HL3T1 cells treatedwith tat but without chloroquine showed similar kinetics of uptake andnuclear localization when assayed by counting the cellular fractions butonly degraded tat was seen on the gel.

The ability of tat to directly enter lymphocytes or monocytes was alsoassessed; tat readily entered both types of cells, as demonstrated bythe high levels of transactivation in cells treated with tat, alone orwith chloroquine. H9 lymphocytes and U937 promonocytes (10⁶ cells)containing an integrated HIV-I LTR-CAT plasmid (H938 and U38 cells,respectively (Felber, B. K. and G. N. Pavlakis, Science 239: 184 (1988))were incubated in RPMI 1640 medium containing 10% fetal bovine serum (1ml in 25 mm wells) at 37° C. (no tat), treated with 5 μg of tat protein(tat) or treated with 5 μg of tat and 100 μm chloroquine (tat+CQ). Cellswere harvested 24 hours after tat treatment and assayed for CAT activity(Gorman, C. M. et al., Mol. Cell Biol. 2: 1044 (1982). HeLa cells (10⁶cells) containing an integrated HIV-1 LTR-CAT plasmid (HL3T1) (Felber,B. K. and G. N. Pavlakis, Science 239: 184 (1988)), were incubated inDulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (1ml in 25 mm wells) and similarly treated with tat protein, or with tatand chloroquine, and assayed for CAT activity. Unacetylated (cm) andacetylated (ac) forms of ¹⁴ C chloramphenicol were separated by thinlayer chromatography.

FIG. 7 shows the results of this assessment of tat entry intolymphocytes and monocytes. High levels of transactivation were seen inall three cell lines. In the HeLa cells, the addition of chloroquineresulted in a significant stimulation of tat activity. However, incontrast to the case with HeLa cells, chloroquine had little effect ontat entry into lymphocytes or monocytes. The chloroquine-independententry into lymphocytes and monocytes may suggest a different mechanismof uptake.

The time course of binding was determined in HeLa (HL3T1) cellscontaining an integrated HIV-I LTR-CAT plasmid (Felber, B. K. and G. N.Pavlakis, Science 239: 184 (1988)). Cells (2×10⁶) were grown toconfluence in 12 well tissue culture plates (12 turn well diameter), andwashed with phosphate buffered saline (PBS). Cells were incubated infresh DMEM with 1 μg tat (1-72) and 100 μM chloroquine at 37° C. fordifferent lengths of time. Following two washes with PBS to remove tat,fresh medium was added and transactivation was measured 24 hours aftertat addition. CAT activity was used as a measure of transactivation.

The results of this analysis are shown in FIG. 8. The basal level ofexpression from the HIV-1 LTR in the absence of tat is shown in the "noTat" lane. Maximal levels of transactivation were observed after a fiveminute exposure to tat. Thus, binding is rapid, and a brief exposure canresult in uptake by cells, as assayed by transactivation.

The time required to observe a response to exogenous tat was determinedin H938 cells. H938 cells were derived from the H9 lymphoid cell line byinfection with a murine sarcoma virus (MSV) retroviral vector. (Felber,B. K. and G. N. Pavlakis, Science 239: 184 (1988)). The integrated MSVvector contains the CAT gene under the control of the HIV-1 LTR, and theneo gene under the control of an SV40 promoter (FIG. 9). H938 cells weremaintained in RPMI 1640 medium supplemented with 10% fetal bovine serum,penicillin (250 U/ml), and streptomycin (250 μg/ml). The cells weretreated with 10 μg/ml of tat protein (amino acids 1-72) in the presenceof 100 μg/ml protamine, and RNA was prepared and analyzed by RNaseprotection. An α-³² P UTP-labeled HIV-1 LTR probe corresponding to a 200bp fragment from -120 to +80 of the viral LTR was prepared by in vitrotranscription. These procedures are further described in Example V.

The results of the RNase protection assay are shown in FIG. 10. Twomajor fragments were protected. The 80 nucleotide fragment is derivedfrom transcripts expressed from the HIV LTR and the 200 nucleotidefragment is derived from transcripts expressed from either the upstreamMSV or SV40 promoters. Transcription from the HIV LTR increased after 15minutes of exposure to tat, and reached a maximum by 2-6 hours. Incontrast, transcription from the upstream MSV and SV40 promoters was notincreased by tat addition, indicating that exogenously added tat retainsspecificity for the HIV promoter. When tat protein is added exogenouslyto cells, there is a significant increase in transcription in 15minutes, indicating that tat can enter cells, become localized to thenucleus, bind to its target site TAR specifically, and promotetranscription within 15 minutes. (The short transcripts, which may bedegradation products from incompletely elongated RNAs, were not affectedby tat.)

Several peptide fragments of tat were tested for their ability tocompete for tat binding and uptake in HL3T1 and H938 cells. In theseexperiments, 0.5×10⁶ H938 cells were pelleted and resuspended in 0.5 mlfresh RPMI 1640 medium. Cells were incubated at 37° C. with 1 μg tat(1-72) and increasing concentrations of peptide. Extracts were preparedafter 24 hours and assayed for CAT activity. Surprisingly, tat 38-58,which contains the basic region of tat, actually enhanced the effect ofexogenous tat and increased transactivation in a concentration dependentmanner. FIG. 11 shows the results of this experiment in the H938 cellline. The data was quantitated by cutting the spots from the TLC platesand counting the associated radioactivity in a scintillation counter.

Protamine (protamine sulfate, Sigma), another basic peptide, was alsoobserved to enhance transactivation by extracellular tat when present ata concentration of 100 μg/ml. However, a smaller tat peptide, containingonly the basic region from 47-58, had no effect on transactivation underthe conditions used. A mixture of two peptides, tat 38-47 and tat 48-58,the products of chymotryptic digestion of tat 38-58, also had no effecton transactivation under these conditions. No enhancement of activity byprotamine was seen when HL3T1 cells were scrape-loaded with tat,suggesting that protamine directly affects the uptake process.

Other cell lines were also tested for tat uptake and transactivationactivity. Jurkat T cells showed significant transactivation when tat wasadded to the medium and showed further transactivation in the presenceof chloroquine. A Vero line (VNHIV-CAT; Mosca, J. D. et al., Nature 325:67-70 (1987)) also showed significant transactivation upon incubation ofcells with tat and chloroquine; no activity was seen with tat alone.However, since the basal expression of CAT was low in this cell line, aseveral fold increase in CAT activity would still have beenundetectable.

To directly follow the entry of tat into live cells, tat was labelledwith rhodamine (TRITC-tat) and its movement was followed by fluorescencemicroscopy. Punctate staining was observed on the surface of HL3T1 andH938 cells immediately after incubation with TRITC-tat, similar to thatseen in receptor-mediated endocytosis. After one hour, clear nuclearstaining was observed in HL3T1 cells. Punctate cytoplasmic staining wasalso observed, suggesting that tat may be localized within endosomes.Incubation at low temperature, which blocks endocytosis, also blockedentry of rhodamine-labeled tat. After six hours, most of the tat was inthe nucleus of HL3T1 cells, but was excluded from the nucleoli.Remarkably, every cell in the culture was labeled with TRITC-tat,indicating that the uptake of exogenous tat is efficient. (Cellularlocalization was also examined in H938 cells, however, since the nucleusconstitutes most of the lymphocytic cell, it was difficult todistinguish nuclear from non-nuclear compartments). When tested fortransactivation, TRITC-tat was found to have the same specific activityas unmodified tat.

Tat-mediated Uptake of a Heterologous Protein

A preliminary assessment of the ability of tat to mediate the uptake ofa molecule of interest was carried out. Additional details of thisanalysis are provided in Example VII. The E2 open reading frame of thebovine papillomavirus-1 (BPV-1; Chen, E. Y. et al., Nature 299: 529-534(1982)) encodes both positive and negative acting transcriptionalregulators (regulatory factors; Sousa, R. et al., Biochim. Biophys. Acta1032: 19-37 (1990); Lambert, P. F et al., J. Virol. 63(7):3151-3154(1989); Lambert, P. F. et al., Cell 50: 69-78 (1987)). A fusion gene wasconstructed in which the HIV-1 tat gene was linked to thecarboxy-terminal region of the E2 open reading frame. The constructwhich encodes the fusion protein, pFTE103 (constructed by Dr. J.Barsoum, Biogen, Inc.), was designed to express a protein comprisingamino acids 1 through 67 of tat at the amino terminus, followed by theC-terminal 105 amino acids of E2 (residues 306 through 410 of BPV-1 E2),which contain the DNA binding domain of the E2 open reading frame (EP0,302,758, Androphy et al., (Feb. 6, 1989); Giri, I. and Yaniv, M., EMBOJ. 7(9):2823-2829 (1988); McBride, A. A. et al., EMBO J. 7(2):533-539(1988); Androphy, E. J. et al., Nature 325: 70-73 (1987)). pFTE103 wasintroduced into E. coli and the TatE2C fusion protein was expressedusing the T7 RNA polymerase expression system as described by Studier etal. (Studier et al., Methods in Enzymology 185: 60-89 (1990)). Thepurified tatE2C fusion protein migrated with an apparent molecularweight of 20,000 to 21,000 daltons on protein gels. Uptake of the TatE2Cfusion protein was tested following introduction into the culture mediumof animal cells.

Uptake of the tat portion of the fusion protein (molecule ofinterest-tat protein conjugate) was assayed by measuring transactivationof a tat-responsive reporter construct integrated into HeLa cells(HeLa.318 cells). The tat-responsive reporter construct (pXB318) presentin HeLa.318 cells contains the human tissue plasminogen activator (tPA)cDNA reporter gene from pTPA114 (Fisher et al. J. Biol. Chem. 260:11223-11230 (1985)) under the control of the HIV-1 long terminal repeat(LTR) from pU3R-III (Sodroski et al. Science 227: 171-173 (1985)). Tatprotein (amino acids 1-72) or the TatE2C fusion protein were added tothe culture medium of HeLa.318 cells in 24 well plates at concentrationsranging from 2.5 nM to 250 nM, in the presence of 100 μM chloroquine,essentially as described (Frankel, A. D. and Pabo, C. O., Cell 55:1189-1193 (1988)). The culture medium was harvested 24 hours later andassayed for tPA activity by the method of Granelli-Piperno and Reich (J.Exp. Med. 148: 223-234 (1978)). Cell numbers were determined and tPAsecretion was expressed as ng/10⁶ cells per day. FIG. 12 shows theresults obtained from a tPA assay of HeLa.318 media 24 hours after theaddition of tat or TatE2C protein to culture medium. In the absence oftat or the TatE2C protein, tPA activity was undetectable (less than 0.1ng/10⁶ cells per day). However, addition of either tat or TatE2C proteinled to an increase in tPA production (FIG. 12). Thus, it appears thattat (residues 1-67) can retain the ability to enter cells when linked toa heterologous protein.

Although transactivation upon addition of the TatE2C protein wassomewhat less efficient than that observed upon addition of tat, theTatE2C fusion protein was also less active than tat in transactivationassays when the proteins were produced intracellularly aftertransfection of the genes into HeLa.318 cells. Thus, it is not clearwhether the apparent reduction in activity is attributable to reduceduptake or reduced activity of the fusion protein produced by E. coli andadded exogenously. It is possible that some tat activity may be lostduring the denaturation and refolding of the TatE2C fusion proteinduring purification.

Uptake of the E2 portion of the conjugate was determined by indirectimmunofluorescence using rabbit polyclonal serum raised against E2-C85(the C-terminal 85 amino acids of the E2 protein produced in E. coli).For indirect immunofluorescence, mouse 3T3 cells were seeded intoLAB-TEK four chamber tissue culture chamber/slides. The next day, TatE2Cfusion protein was added at 250 nM to the culture medium, in thepresence of 100 μM chloroquine. Six hours later, immunofluorescence wasperformed as described in Example VII.

While only very faint background fluorescence was seen when E2.C85protein was added to cells (at the same concentration and in thepresence of 100 μM chloroquine), addition of the TatE2C fusion proteinled to very intense fluorescence in all cells observed. These cellsdisplayed fluorescence on the plasma membrane, in the cytosol and innuclei. The staining was present in bright patches rather than evenlydispersed throughout the cells. The amount of E2 fluorescence obtainedfollowing addition of TatE2C protein to culture medium was far greaterthan the immunofluorescence observed when a TatE2C gene was expressed inthese same cells. These data indicate that the tat protein is capable ofefficiently carrying a heterologous protein present as part of amolecule of interest-tat conjugate into cells.

In order that the invention described herein may be more fullyunderstood, the following examples are set forth. It should beunderstood that these examples are for illustrative purposes only andare not to be construed as limiting this invention in any manner.Throughout these examples, all molecular cloning reactions were carriedout according to methods in J. Sambrook et al., Molecular Cloning: ALaboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory (1989),except where otherwise noted.

EXAMPLE I Bacterial Expression and Purification of Tat

Two plasmids were constructed to produce the tat protein in E. coli; oneexpresses amino acids 1-86 (the entire coding sequence) and the otherexpresses the first coding exon of tat (residues 1-72). It is known thatthe second exon is not required for activity (Cullen, B. R., Cell 46:973-982 (1986); Muesing, M. A., et al., Cell 48: 691-701 (1987);Sodroski, J., et al., Science 229: 74-77 1985); Frankel, A. D., et al.,Proc. Natl. Acad. Sci. USA 86: 7397-7401 (1989)). Synthetic tat geneswere constructed and ligated into the NdeI site of pET-3a, a plasmidthat uses a strong bacteriophage T7 promoter to express cloned genes(Studier, F. W. and B. M. Moffat, J. Mol. Biol. 189: 113-130 (1986);Rosenberg, A. H., et al., Gene 56: 125-135 (1987)). The resultingplasmids, ptat72 and ptat86, express tat (residues 1-72 or 1-86,respectively) as 1%-5% of total E. coli protein. Both proteins gavesimilar results in all experiments. BL21(DE3) cells were used forexpression and these cells also contained a plasmid expressing the T7lysozyme gene to inhibit any T7 RNA polymerase expressed prior toinduction (F. W. Studier, personal communication). Tat was induced withisopropyl β-D-thiogalactopyranoside (IPTG) (Studier, F. W. and B. M.Moffat, J. Mol. Biol. 189: 113-130 (1986)) and purified essentially asdescribed (Frankel, A. D., et al., Science 240: 70-73 (1988)) exceptthat tat was extracted from the polyethyleneimine pellet with 10%ammonium sulfate instead of 700 mM KCl, and the S-Sepharosechromatography was eliminated.

EXAMPLE II Synthetic Tat Peptides

Syntheses were performed using Fmoc chemistry on a Milligen/Biosearchmodel 9600 peptide synthesizer with a peptide amide linker-norleucine4-methylbenzhydrylamine (PAL-NIe-MBHA) polystyrene resin(Milligen/Biosearch; 0.5 g). Thebenzotriazolyloxytris(dimethylamino)phosphoniumhexafluorophosphate/l-hydroxybenzotriazole (BOP/HOBt) coupling method(Hudson, D., J. Org. Chem. 53: 617-624 (1988)) was used with couplingtimes of 1-4 hours and with double coupling of His-33. Protecting groupswere t-butyl ester (for Glu and Asp),2,2,5,7,8-pentamethylchroman-6-sulfonyl (Arg), t-butyloxycarbonyl (Lys),trityl (His and Cys), t-butyl (Ser, Thr, and Tyr), and trimethoxybenzyl(Asn and Gin). All peptides were synthesized as their C-terminal amides.After synthesis was completed, protecting groups were removed and thepeptide chains were cleaved from the resin with trifluoroaceticacid/ethanedithiol/thioanisole/anisole (90:3:5:2, vol/vol). The mixturewas filtered and the products were obtained by addition of coldanhydrous diethyl ether to the filtrate. The precipitate was collectedby filtration, thoroughly washed with ether and dried.

Peptides were treated with 0.5M dithiothreitol at 37° C. for 30 minutesto ensure complete reduction of the cysteines and were purified on a C₄HPLC column (Vydac) using an acetonitrile gradient in 0.1%trifluoroacetic acid. Amino acid composition was determined byhydrolysis in 6M HCl containing 0.5% phenol at 100° C. and analysis on aLKB Alpha Plus analyzer. Peptide purity (>90%) was determined by HPLCusing an acetonitrile gradient of <0.5% per minute.

EXAMPLE III Uptake of ¹²⁵ I-Labeled Tat

Tat (residues 1-72) was labeled with ¹²⁵ I by treating 500 μg of proteinwith 0.5 mCi ¹²⁵ I and IODO-BEADS (Pierce) in 0.1M Tris-HCl (pH 7.5) atroom temperature for 5 minutes. The sample was dialyzed to removeunreacted ¹²⁵ I. The specific activity was approximately 10⁶ cpm/μgprotein. HL3T1 cells (2×10⁶ cells per dish) were treated with 5 μgradioactive tat in the presence or absence of 100 μM chloroquine. Mediumwas removed at various times, cells were washed with PBS and EDTA, andcells were trypsinized for 10 minutes. Pancreatic trypsin inhibitor wasadded (5 μg/ml), cells were chilled to 4° C., centrifuged at 100×g, andthe supernatant was saved. The cell pellet was washed twice withserum-free DMEM, once with PBS and nuclei were isolated by lysis in 0.5%NP-40 as described (Ausubel, F. M. et al., Current Protocols inMolecular Biology (New York: John Wiley and Sons, 1987)). ¹²⁵ I wascounted using an LKB gamma counter.

EXAMPLE IV Chloroquine Stimulated Tat Uptake

The parameters of chloroquine-stimulated tat activity were studied inmore detail. FIG. 6A shows that the concentration dependence ofchloroquine is a rather sharp dose response with maximum transactivationobserved at 100 μM chloroquine. This concentration is typically used toraise vacuolar pH (Mellman, I. et al., Annu. Rev. Biochem. 55: 663-700(1986)).

The time course of tat transactivation in the presence of chloroquineshowed a plateau after 24 hours (FIG. 6B), and transactivation in thepresence of chloroquine increased with increasing tat concentration(FIG. 6C). Transactivation was detectable with tat concentrations as lowas 1 nM.

Controls were done to determine whether transactivation was dependent onan intact TAR site, to determine whether a heterologous promoter couldbe stimulated by tat, and to determine whether any of the effects seenwith chloroquine occurred when tat was produced intracellularly. Aftertransient transfection of HeLa cells with an HIV-LTR plasmid (p-167/+80;Rosen, C. A. et al., Cell 41: 813-823 (1985)), high levels oftransactivation were seen when tat was introduced by cotransfection witha tat expression plasmid (pSV2tat72), by scrape-loading purified tat, orby treatment with tat and chloroquine. However, expression from theHIV-LTR containing a mutant TAR site (p-167/+21; Rosen, C. A. et al.,Cell 41: 813-823 (1985)) or from the SV40 early promoter (pSV2-CAT;Gorman, C. M. et al., Mol. Cell. Biol. 2: 1044-1051 (1982)) was notstimulated when tat was introduced by these methods. Thus, introducingtat by scrape-loading or by uptake with chloroquine appears totransactivate the HIV-LTR by the same mechanism that occurs when tat isproduced intracellularly. Chloroquine had no effect when tat wasproduced intracellularly; chloroquine treatment of HL3T1 cellstransiently transfected with pSV2tat72 showed no additionaltransactivation.

EXAMPLE V RNA Isolation and Analysis

For the RNase protection experiment, total RNA was isolated by the hotacidic phenol method (Queen, C. and D. Baltimore, Cell 33: 741-748(1983)). HIV-1-specific probes for all hybridizations were prepared byin vitro transcription (with α-³² P UTP) of an EcoRV-linearized plasmidcontaining the EcoRV (-120) to HindIII (+80) fragment of the viral LTR(cloned into the plasmid sp73; Promega). RNA probes were purified onSephadex G-50 spin columns (Boehringer-Mannheim).

RNase protection experiments were performed as described (Ausubel, F. M.et al., Current Protocols in Molecular Biology (New York: John Wiley andSons, 1987)). Twenty μg of cellular RNA were hybridized overnight with5×10⁵ cpm of the RNA probe at 38° C. in 40 μl of 80% formamide, 40 mMPIPES (pH 6.7), 200 mM NaCl, 1 mM EDTA. Single-stranded RNA was digestedwith RNase A (10 μg/ml) and RNase Ti (45 U/ml) (Boehringer-Mannheim) in400 μl of 10 mM Tris-HCl (pH 7.5), 300 mM NaCl, 5 mM EDTA for 1 hour atroom temperature. Protected fragments were analyzed by electrophoresison 6% polyacrylamide-7M urea sequencing gels. Protected RNAs werevisualized by autoradiography with intensifying screens and werequantitated using a Betascope 603 (Betagen).

EXAMPLE VI Localization of Tat by Fluorescence

Purified Tat protein was labeled at lysine residues with tetramethylrhodamine isothiocyanate (TRITC) by incubating 200 μg of tat (aminoacids 1-72) in 0.1M Na₂ CO₃ pH 9.0 with 5 μg of TRITC dissolved in 5 μldimethylsulfoxide (DMSO), for 8 hours at 4° C. Unreacted TRITC wasquenched with 50 mM NH₄ Cl. The pH was lowered to 7.0 with HCl andrhodamine-labeled Tat was purified from free TRITC by dialysis against50 mM Tris, pH 7, 1 mM DTT.

HL3T1 cells were grown on glass coverslips and incubated for variouslengths of time with rhodamine-conjugated tat (TRITC-Tat) in DMEM. H938cells in suspension were incubated with rhodamine-conjugated tat inRPMI. Cells were washed three times with phosphate buffered saline andviewed live on a Zeiss Axiophot fluorescence microscope.

EXAMPLE VII Uptake of TatE2C Fusion Protein

Cell Lines

The mouse embryo fibroblast cell line Balb/c 3T3 (clone A31; Aaronsonand Todaro, J. Cell Physiol. 72: 141-148 (1968)) was obtained from theAmerican Type Culture Collection. HeLa cells were obtained from Dr. AlanFrankel (Whitehead Institute, MIT). Both cell lines were propagated inDulbecco's minimal essential medium (GIBCO) supplemented with 10% donorcalf serum (Hazelton) and 4 mM glutamine (Whittaker). Cells were grownin a 5.5% CO₂ incubator at 37° C. Passaging of cells was performed bywashing with phosphate-buffered saline and treating with trypsin (bothGIBCO) to remove cells from plates followed by addition of culturemedium and dilution of cells into plates containing fresh culturemedium.

The HeLa cell line containing a tat-responsive reporter construct(HeLa.318) was generated by the introduction and stable selection ofplasmid pXB318 (described below) by electroporation as described by Chuet al. (Chu et al., Nucleic Acids Res. 15: 1311-1326 (1987)). pXB318 DNAwas electroporated together with the selectable marker pSV2-neo(Southern, E. M. and Berg, P., J. Mol. Appl. Genet. 1: 327-341 (1982)).Stable transfectants were selected in the presence of G418 (Southern, E.M. and Berg, P., J. Mol. Appl. Genet. 1: 327-341 (1982)), and thepresence of pXB318 DNA was confirmed by Southern blot hybridizationanalysis (Southern, E. M., J. Mol. Biol. 98: 503-517 (1975)).

Vector Constructions

All molecular cloning reactions were carried out by methods described byManiatis et al. (Maniatis, T., Fritsch, E. F., and Sambrook, J.,Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory,NY (1982)), using enzymes obtained from New England Biolabs (Beverly,Mass.).

The TatE2C fusion protein (protein TatE2C), in which HIV tat was fusedto the carboxy terminal portion of BPV-1 E2, was expressed from thebacterial expression plasmid pFTE103. This plasmid was derived fromptat72 (see Example I) by insertion of a StyI-SpeI fragment which wasisolated from vector pCO-E2 (Hawley-Nelson et al., EMBO J. 7: 525-531(1988)) and which encodes the C-terminal portion of the E2 protein. Foursynthetic deoxyoligonucleotides were used in the construction describedbelow in detail.

The plasmid ptat72 was cleaved with the restriction endonucleases NdeIand BamHI, releasing the tat encoding portion of the vector. The 4603base pair (bp) vector fragment was purified by agarose gelelectrophoresis, and a 169 base pair (bp) NdeI-AatII fragment of the tatencoding fragment was isolated. The 3' portion of the E2C codingsequence was isolated as a 375 bp StyI-SpeI fragment from pCO-E2(Hawley-Nelson et al., EMBO J. 7: 525-531 (1988); obtained from Dr.Elliot Androphy, Tufts University/New England Medical Center Hospitals).The E2C fragment was connected to the tat fragment and to the expressionvector by use of two pairs of complementary deoxyoligonucleotides(synthesized according to standard procedures using an AppliedBiosystems 380A DNA Synthesizer). One complementary pair ofoligonucleotides was designed to join the AatII overhang of the tatfragment to the StyI overhang of the E2C fragment. The complementarypair of oligonucleotides consisted of oligonucleotides 374-3 (SEQ IDNO:64) and 374-4 (SEQ ID NO:65). A second pair of complementaryoligonucleotides was designed to link the SpeI overhang of the E2Cfragment to the BamHI overhang of the 4603 bp vector backbone isolatedfrom ptat72. The second pair of complementary oligonucleotides consistedof oligonucleotides 374-5 (SEQ ID NO:66) and 374-6 (SEQ ID NO:67). Thetat fragment, the E2C fragment and the two pairs of oligos were insertedinto the 4603 ptat72 vector backbone to create pFTE103. The resultingfusion gene is designed to express a protein comprising amino acids 1through 67 of tat at the amino terminus followed by the C-terminal 105amino acids of E2 (residues 306 through 410 of BPV-1 E2).

The tat-responsive reporter construct pXB318 was constructed in threesteps. The starting plasmid was pBG312 (Cate et al., Cell 45: 685-698(1986)). Two oligodeoxynucleotides, with sequences consisting of (SEQ IDNO:68) and (SEQ ID NO:69), were synthesized, which when annealed have anAatII-compatible overhang at the 5' end and an EcoRI-compatible overhangat the 3' end, and form a polylinker with internal XhoI, HindIII andBamHI restriction sites. Plasmid pBG312 was cleaved with AatII and EcoRIto remove the promoter, and the above polylinker was inserted into thevector to form the promoterless vector pXB100. The HIV-1 long terminalrepeat (LTR) from pU3R-III (Sodroski et al. Science 227: 171-173 (1985))was excised as a XhoI-HindIII fragment and was inserted into XhoI andHindIII sites of the polylinker of pXB100 to create pXB301. The humantissue plasminogen activator (tPA) cDNA reporter was excised as a BamHIfragment from pTPA114 (Fisher et al., J. Biol. Chem. 260: 11223-11230(1985)) and inserted into the BamHI site of pXB301 to create pXB318.

Expression and Purification of TatE2C

The TatE2C fusion protein was expressed in E. coli using the vectorpFTE103 and the T7 RNA polymerase expression system precisely asdescribed by Studier et al. (Studier et al., Methods in Enzymology 185:60-89 (1990)). Virtually all of the TatE2C protein was found in theinsoluble fraction. The following purification was performed:

1. E. coli were pelleted, resuspended in ten packed cell volumes of 25mM Tris-HCl pH 7.5, 1 mM EDTA, 10 mM DTT, and 1 mM PMSF and lysed withtwo passages through a French press.

2. The membrane fraction was pelleted by centrifugation at 10,000 rpmfor 30 minutes.

3. This membrane fraction was resuspended in 6M urea.

4. Solid guanidine-HCl was added to a final concentration of 6M and DTTwas added to a final concentration of 10 mM.

5. After 30 minutes at 37° C., the solution was clarified bycentrifugation at 10,000 rpm for 30 minutes.

6. The sample was loaded onto an A.5 agarose gel filtration column in 6Mguanidine-HCl, 50 mM sodium phosphate pH 5.4, and 10 mM DTT.

7. TatE2C-containing fractions were loaded onto a C₁₈ reverse phase HPLCcolumn and eluted with a gradient of 0-75% acetonitrile in 0.1%trifluoroacetic acid.

TatE2C protein appeared in a single peak. On protein gels, the TatE2Cfusion protein migrated with an apparent molecular weight of 20,000 to21,000 daltons.

Assay of TatE2C Uptake by Tat Activity

Uptake was detected either as tat activity (activation of atat-dependent reporter in HeLa.318) or by indirect immunofluorescenceusing anti-E2 antibodies. Tat activity was determined by adding tatprotein (amino acids 1-72) or TatE2C fusion protein at 2.5-250 nM alongwith chloroquine at 0.1 mM to the culture medium of HeLa.318 cells in 24well plates essentially by the method of Frankel and Pabo (Frankel, A.D. and Pabo, C. O., Cell 55: 1189-1193 (1988)). The culture medium washarvested 24 hours later and assayed for tPA activity by the method ofGranelli-Piperno and Reich (J. Exp. Med. 148: 223-234 (1978)). Cellnumbers were determined and tPA secretion was expressed as ng/10⁶ cellsper day. tPA secretion was undetectable in the absence of added tat orTatE2C protein (less than 0.1 ng/10⁶ cells per day).

Assay of TatE2C Uptake by E2-Specific Immunofluorescence

For indirect immunofluorescence, mouse 3T3 cells were seeded intoLAB-TEK four chamber tissue culture chamber/slides. On the next day,TatE2C protein and chloroquine were added to the culture medium to finalconcentrations of 250 nM and 0.1 mM, respectively. Six hours later,immunofluorescence was performed as follows:

1. Medium was removed and wells were washed twice withphosphate-buffered saline (PBS).

2. Cells were fixed by treatment with 3.5% formaldehyde for 10 minutesat room temperature.

3. Cells were permeabilized in 0.2% Triton X-100/2% bovine serum albumin(BSA) in PBS with 1 mM MgCl₂ /0.1 mM CaCl₂ (PBS+) for 5 minutes at roomtemperature.

4. Cells were blocked by treatment with whole goat serum (Cappel#5506-1380) at a 1:30 dilution in PBS+/2% BSA for one hour at 4° C.

5. The primary antibody was an affinity purified rabbit polyclonal whichhad been raised by injection of purified protein E2.C85 (in this casethe carboxy terminal 85 amino acids expressed in bacteria using the T7polymerase expression system) into a rabbit, followed by purification bypassage of the bleed over an E2 affinity column.

This antibody was added to the wells at a 1:100 dilution in PBS+/2% BSAfor one hour at 4° C.

6. The secondary antibody was a rhodamine conjugated goat anti-rabbitIgG (Cappel #2212-0081). This antibody was added at a 1:100 dilution inPBS+/0.2% BSA for 30 minutes at 4° C.

7. Wells were washed three times with PBS+/0.2% Tween-20/2% BSA.

8. Slides were mounted in 50% glycerol in PBS and viewed with afluorescent microscope with a rhodamine filter.

As a control, purified E2C protein (the carboxy terminal 85 amino acidswhich were found to be recognized by the polyclonal antibodypreparation) was added to wells in the same manner as the TatE2C fusionprotein.

EXAMPLE 1 Production and Purification of Transport Polypeptides

Recombinant DNA

Plasmid pTat72 was a starting clone for bacterial production oftat-derived transport polypeptides and construction of genes encodingtransport polypeptide-cargo protein fusions. We obtained plasmid pTat72(described in Frankel and Pabo, supra and in Example I, above) from AlanFrankel (The Whitehead Institute for Biomedical Research, Cambridge,Mass.). Plasmid pTat72, was derived from the pET-3a expression vector ofF. W. Studier et al. ("Use of T7 RNA Polymerase to Direct Expression ofCloned Genes", Methods Enzymol., 185, pp. 60-90 (1990)) by insertion ofa synthetic gene encoding amino acids 1 to 72 of HIV-1 tat. The tatcoding region employs E.coli codon usage and is driven by thebacteriophage T7 polymerase promoter inducible with isopropylbeta-D-thiogalactopyranoside ("IPTG"). Tat protein constituted 5% oftotal E.coli protein after IPTG induction.

Purification of Tat1-72 from Bacteria

We suspended E.coli expressing tat1-72 protein in 10 volumes of 25 mMTris-HCl (pH 7.5), 1 mM EDTA. We lysed the cells in a French press andremoved the insoluble debris by centrifugation at 10,000×g for 1 hour.We loaded the supernatant onto a Q Sepharose Fast Flow (Pharmacia LKB,Piscataway, N.J.) ion exchange column (20 ml resin/60 ml lysate). Wetreated the flow-through fraction with 0.5M NaCl, which caused the tatprotein to precipitate. We collected the salt-precipitated protein bycentrifugation at 35,000 rpm, in a 50.2 rotor, for 1 hour. We dissolvedthe pelleted precipitate in 6M guanidine-HCl and clarified the solutionby centrifugation at 35,000 rpm, in a 50.2 rotor, for 1 hour. We loadedthe clarified sample onto an A.5 agarose gel filtration columnequilibrated with 6M guanidine-HCl, 50 mM sodium phosphate (pH 5.4), 10mM DTT, and then eluted the sample with the same buffer. We loaded thetat protein-containing gel filtration fractions onto a C₄ reverse phaseHPLC column and eluted with a gradient of 0-75% acetonitrile, 0.1%trifluoroacetic acid. Using this procedure, we produced about 20 mg oftat1-72 protein per liter of E.coli culture (assuming 6 g of cells perliter). This represented an overall yield of about 50%.

Upon SDS-PAGE analysis, the tat1-72 polypeptide migrated as a singleband of 10 kD. The purified tat1-72 polypeptide was active in anuptake/transactivation assay. We added the polypeptide to the culturemedium of human hepatoma cells containing a tat-responsive tissueplasminogen activator ("tPA") reporter gene. In the presence of 0.1 mMchloroquine, the purified tat1-72 protein (100 ng/ml) induced tPAexpression approximately 150-fold.

Chemical Synthesis of Transport Polypeptides

For chemical synthesis of the various transport polypeptides, we used acommercially-available, automated system (Applied Biosystems Model 430Asynthesizer) and followed the system manufacturer's recommendedprocedures. We removed blocking groups by HF treatment and isolated thesynthetic polypeptides by conventional reverse phase HPLC methods. Theintegrity of all synthetic polypeptides was confirmed by massspectrometer analysis.

EXAMPLE 2 β-Galactosidase Conjugates

Chemical Cross-Linking with SMCC

For acetylation of β-galactosidase (to block cysteine sulfhydryl groups)we dissolved 6.4 mg of commercially obtained β-galactosidase (PierceChem. Co., cat. no. 32101G) in 200 μl of 50 mM phosphate buffer (pH7.5). To the 200 μl of β-galactosidase solution, we added 10 μl ofiodoacetic acid, prepared by dissolving 30 mg of iodoacetic acid in 4 mlof 50 mM phosphate buffer (pH 7.5). (In subsequent experiments we foundiodoacetamide to be a preferable substitute for iodoacetic acid.) Weallowed the reaction to proceed for 60 minutes at room temperature. Wethen separated the acetylated β-galactosidase from the unreactediodoacetic acid by loading the reaction (Pharmacia) mixture on a smallG-25 (Pharmacia LKB, Piscataway, N.J.) gel filtration column andcollecting the void volume.

Prior to SMCC activation of the amine groups of the acetylatedβ-galactosidase, we concentrated 2 ml of the enzyme collected from theG-25 column to 0.3 ml in a Centricon 10 (Amicon, Danvers, Mass.)ultrafiltration apparatus. To the concentrated acetylatedβ-galactosidase, we added 19 μg of sulfo-SMCC (Pierce Chem. Co., cat.no. 22322G) dissolved in 15 μl of dimethylformamide ("DMF"). We allowedthe reaction to proceed for 30 minutes at room temperature. We thenseparated the β-galactosidase-SMCC from the DMF and unreacted SMCC bypassage over a small G-25 gel filtration column.

For chemical cross-linking of transport polypeptides to β-galactosidase,we mixed the solution of β-galactosidase-SMCC with 100 μg of transportpolypeptide (tat1-72, tat37-72, tat38-58GGC, tat37-58, tat47-58GGC ortatCGG47-58) dissolved in 200 μl of 50 mM phosphate buffer (pH 7.5). Weallowed the reaction to proceed for 60 minutes at room temperature. Wethen isolated the transport polypeptide-β-galactosidase conjugate byloading the reaction mixture on an S-200HR gel filtration column andcollecting the void volume.

The transport polypeptide-β-galactosidase conjugate thus obtainedyielded positive results when assayed for tat in conventional Westernblot and ELISA analyses performed with rabbit anti-tat polyclonalantibodies. For a general discussion of Western blot and ELISA analysis,see E. Harlow and D. Lane, Antibodies: A Laboratory Manual, Cold SpringHarbor Laboratory (1988). Gel filtration analysis with Superose 6(Pharmacia LKB, Piscataway, N.J.) indicated the transportpolypeptide-β-galactosidase conjugate to have a molecular weight ofabout 540,000 daltons. Specific activity of the transportpolypeptide-β-galactosidase conjugate was 52% of the specific activityof the β-galactosidase starting material, when assayed witho-nitrophenyl-β-D-galactopyranoside ("ONPG"). The ONPG assay procedureis described in detail at pages 16.66-16.67 of Sambrook et al. (supra).

Cellular Uptake of β-Galactosidase Conjugates

We added the conjugates to the medium of HeLa cells (ATCC no. CCL2) at20 μg/ml, in the presence or absence of 100 μM chloroquine. We incubatedthe cells for 4-18 hours at 37° C./5.5% CO₂. We fixed the cells with 2%formaldehyde, 0.2% glutaraldehyde in phosphate-buffered saline ("PBS")for 5 minutes at 4° C. We then washed the cells three times with 2 mMMgCl₂ in PBS, and stained them with X-gal, at 37° C. X-gal is acolorless β-galactosidase substrate (5-bromo-4-chloro-3-indolylD-galactoside) that yields a blue product upon cleavage byβ-galactosidase. Our X-gal staining solution contained 1 mg of X-gal(Bio-Rad, Richmond, Calif., cat. no. 170-3455) per ml of PBS containing5 mM potassium ferricyanide, 5 mM potassium ferrocyanide and 2 mM MgCl₂.

We subjected the stained cells to microscopic examination atmagnifications up to 400×. Such microscopic examination revealed nuclearstaining, as well as cytoplasmic staining.

The cells to which the tat37-72-β-galactosidase conjugate ortat1-72-β-galactosidase conjugate was added stained dark blue.β-galactosidase activity could be seen after a development time as shortas 15 minutes. For comparison, it should be noted that stain developmenttime of at least 6 hours is normally required when β-galactosidaseactivity is introduced into cells by means of transfection of theβ-galactosidase gene. Nuclear staining was visible in the absence ofchloroquine, although the nuclear staining intensity was slightlygreater in chloroquine-treated cells. Control cells treated withunconjugated β-galactosidase showed no detectable staining.

Cleavable Conjugation by Direct Disulfide

Each β-galactosidase tetramer has 12 cysteine residues that may be usedfor direct disulfide linkage to a transport polypeptide cysteineresidue. To reduce and then protect the sulfhydryl of tat37-72, wedissolved 1.8 mg (411 nmoles) of tat37-72 in 1 ml of 50 mM sodiumphosphate (pH 8.0), 150 mM NaCl, 2 mM EDTA, and applied the solution toa Reduce-Imm column (Pierce Chem. Co., Rockford, Ill.). After 30 minutesat room temperature, we eluted the tat37-72 from the column with 1 mlaliquots of the same buffer, into tubes containing 0.1 ml of 10 mM5,5'-dithio-bis(2-nitrobenzoic acid) ("DTNB"). We left the reducedtat37-72 polypeptide in the presence of the DTNB for 3 hours. We thenremoved the unreacted DTNB from the tat37-72-TNB by gel filtration on a9 ml Sephadex G-10 column (Pharmacia LKB, Piscataway, N.J.). Wedissolved 5 mg β-galactosidase in 0.5 ml of buffer and desalted it on a9 ml Sephadex G-25 column (Pharmacia LKB, Piscataway, N.J.), to obtain3.8 mg of β-galactosidase/ml buffer. We mixed 0.5 ml aliquots ofdesalted β-galactosidase solution with 0.25 or 0.5 ml of thetat37-72-TNB preparation, and allowed the direct disulfide cross-linkingreaction to proceed at room temperature for 30 minutes. We removed theunreacted tat37-72-TNB from the β-galactosidase conjugate by gelfiltration on a 9 ml Sephacryl S-200 column. We monitored the extent ofthe cross-linking reaction indirectly, by measuring absorbance at 412 nmdue to the released TNB. The direct disulfide conjugates thus producedwere taken up into cells (data not shown).

Cleavable Conjugation with SPDP

We used the heterobifunctional cross-linking reagent ("SPDP"), whichcontains a cleavable disulfide bond, to form a cross-link between: (1)the primary amine groups of β-galactosidase and the cysteine sulfhydrylsof tat1-72 (metabolically labelled with ³⁵ S); or (2) the primary aminegroups of rhodamine-labelled β-galactosidase and the amino terminalcysteine sulfhydryl of tat37-72.

For the tat1-72 conjugation, we dissolved 5 mg of β-galactosidase in 0.5ml of 50 mM sodium phosphate (pH 7.5), 150 mM NaCl, 2 mM MgCl₂, anddesalted the β-galactosidase on a 9 ml Sephadex G-25 column (PharmaciaLKB, Piscataway, N.J.). We treated the desalted β-galactosidase with an88-fold molar excess of iodoacetamide at room temperature for 2 hours,to block free sulfhydryl groups. After removing the unreactediodoacetamide by gel filtration, we treated the blocked β-galactosidasewith a 10-fold molar excess of SPDP at room temperature. After 2 hours,we exchanged the buffer, by ultrafiltration (Ultrafree 30, Millipore,Bedford, Mass.). We then added a 4-fold molar excess of labelledtat1-72, and allowed the cross-linking reaction to proceed overnight, atroom temperature. We removed the unreacted tat1-72 by gel filtration ona 9 ml Sephacryl S-200 column. Using the known specific activity of thelabelled tat1-72, we calculated that there were 1.1 tat1-72 polypeptidescross-linked per β-galactosidase tetramer. Using the ONPG assay, wefound that the conjugated β-galactosidase retained 100% of its enzymaticactivity. Using measurement of cell-incorporated radioactivity and X-galstaining, we demonstrated uptake of the conjugate into cultured HeLacells.

For the tat37-72 conjugation, our procedure was as described in thepreceding paragraph, except that we labelled the β-galactosidase with a5:1 molar ratio of rhodamine maleimide at room temperature for 1 hour,prior to the iodoacetamide treatment (100:1 iodoacetamide molar excess).In the cross-linking reaction, we used an SPDP ratio of 20:1, and atat37-72 ratio of 10:1. We estimated the conjugated product to haveabout 5 rhodamine molecules (according to UV absorbance) and about 2tat37-72 moieties (according to gel filtration) per β-galactosidasetetramer. The conjugate from this procedure retained about 35% of theinitial G-galactosidase enzymatic activity. Using X-gal staining andrhodamine fluorescence, we demonstrated that the SPDP conjugate wastaken up into cultured HeLa cells.

EXAMPLE 3 Animal Studies with β-Galactosidase Conjugates

For conjugate half-life determination and biodistribution analysis, weinjected either 200 μg of SMCC-β-galactosidase (control) ortat1-72-β-galactosidase intravenously ("IV") into the tail veins ofBalb/c mice (Jackson Laboratories), with and without chloroquine. Wecollected blood samples at intervals up to 30 minutes. After 30 minutes,we sacrificed the animals and removed organs and tissues forhistochemical analysis.

We measured β-galactosidase activity in blood samples by the ONPG assay.The ONPG assay procedure is described in detail at pages 16.66-16.67 ofSambrook et al. (supra). β-galactosidase and tat1-72-β-galactosidasewere rapidly cleared from the bloodstream. We estimated their half-livesat 3-6 minutes. These experimental comparisons indicated that attachmentof the tat1-72 transport polypeptide has little or no effect on theclearance rate of β-galactosidase from the blood.

To detect cellular uptake of the transport polypeptide-β-galactosidaseconjugates, we prepared thin frozen tissue sections from sacrificedanimals (above), carried out fixation as described in Example 2 (above),and subjected them to a standard X-gal staining procedure. Liver, spleenand heart stained intensely. Lung, and skeletal muscle stained lessintensely. Brain, pancreas and kidney showed no detectable staining.High power microscopic examination revealed strong cellular, and in somecases, nuclear staining of what appeared to be endothelial cellssurrounding the blood supply to the tissues.

EXAMPLE 4 Cellular Uptake Tests with β-Galactosidase-Polyarginine andβ-Galactosidase-Polylysine Conjugates

To compare the effectiveness of simple basic amino acid polymers withthe effectiveness of our tat-derived transport polypeptides, weconjugated commercially available polyarginine (Sigma Chem Co., St.Louis, Mo., cat. no. P-4663) and polylysine (Sigma cat. no. P-2658) toβ-galactosidase, as described in Example 2, above. We added theconjugates to the medium of HeLa cells at 1-30 μg/ml, with and withoutchloroquine. Following incubation with the conjugates, we fixed, stainedand microscopically examined the cells as described in Example 2, above.

The polylysine-β-galactosidase conjugate gave low levels of surfacestaining and no nuclear staining. The polyarginine-β-galactosidaseconjugate gave intense overall staining, but showed less nuclear stainthan the tat1-72-β-galactosidase and tat37-72-β-galactosidaseconjugates. To distinguish between cell surface binding and actualinternalization of the polyarginine-β-galactosidase conjugate, wetreated the cells with trypsin, a protease, prior to the fixing andstaining procedures. Trypsin treatment eliminated most of the X-galstaining of polyarginine-1-galactosidase treated cells, indicating thatthe polyarginine-β-galactosidase conjugate was bound to the outsidesurfaces of the cells rather than actually internalized. In contrast,cells exposed to the tat1-72 or 37-72-β-galactosidase conjugates staineddespite trypsin treatment, indicating that the β-galactosidase cargo wasinside the cells and thus protected from trypsin digestion. Controlcells treated with unconjugated β-galactosidase showed no detectablestaining.

EXAMPLE 5 Horseradish Peroxidase Conjugates

Chemical Cross-Linking

To produce tat1-72-HRP and tat37-72-HRP conjugates, we used acommercially-available HRP coupling kit (Immunopure maleimide activatedHRP, Pierce Chem. Co., cat. no. 31498G). The HRP supplied in the kit isin a form that is selectively reactive toward free --SH groups.(Cysteine is the only one of the 20 protein amino acids having a free--SH group.) In a transport polypeptide-HRP conjugation experimentinvolving tat1-72, we produced the tat1-72 starting material in E.coliand purified it by HPLC, as described in Example 1, above. Welyophilized 200 μg of the purified tat1-72 (which was dissolved inTFA/acetonitrile) and redissolved it in 100 μl of 100 mM HEPES buffer(pH7.5), 0.5 mM EDTA. We added 50 μl of the tat1-72 or tat37-72 solutionto 50 μl of Immunopure HRP (750 μg of the enzyme) in 250 mMtriethanolamine (pH 8.2). We allowed the reaction to proceed for 80minutes, at room temperature. Under these conditions, approximately 70%of the HRP was chemically linked to tat1-72 molecules. We monitored theextent of the linking reaction by SDS-PAGE analysis.

Cellular Uptake of HRP Conjugates

We added the conjugates to the medium of HeLa cells at 20 μg/ml, in thepresence or absence of 100 μM chloroquine. We incubated the cells for4-18 hours at 37° C./5.5% CO₂. We developed the HRP stain using4-chloro-1-naphthol (Bio-Rad, Richmond, Calif., cat. no. 170-6431) andhydrogen peroxide HRP substrate. In subsequent experiments, wesubstituted diaminobenzidine (Sigma Chem. Co., St. Louis, Mo.) for4-chloro-1-naphthol.

Cells to which we added transport polypeptide-HRP conjugates displayedcell-associated HRP activity. Short time periods of conjugate exposureresulted in staining patterns which appeared punctate, probablyreflecting HRP in endocytic vesicles. Following longer incubations, weobserved diffuse nuclear and cytoplasmic staining. Control cells treatedwith unconjugated HRP showed no detectable staining.

EXAMPLE 6 PE ADP Ribosylation Domain Conjugates

We cloned and expressed in E.coli the Pseudomonas exotoxin ("PE") bothin its full length form and in the form of its ADP ribosylation domain.We produced transport polypeptide-PE conjugates both by genetic fusionand chemical cross-linking.

Plasmid Construction

To construct plasmid pTat70(ApaI), we inserted a unique ApaI site intothe tat open reading frame by digesting pTat72 with BamH1 and EcoR1, andinserting a double-stranded linker consisting of the following syntheticoligonucleotides:

GATCCCAGAC CCACCAGGTT TCTCTGTCGG GCCCTTAAG (SEQ ID NO:8)

AATTCTTAAG GGCCCGACAG AGAAACCTGG TGGGTCTGG (SEQ ID NO:9).

The linker replaced the C-terminus of tat, LysGlnStop, with GlyProStop.The linker also added a unique ApaI site suitable for in-frame fusion ofthe tat sequence with the PE ADP ribosylation domain-encoding sequences,by means of the naturally-occurring ApaI site in the PE sequence. Toconstruct plasmid pTat70PE (SEQ ID NO:10), we removed an ApaI-EcoRIfragment encoding the PE ADP ribosylation domain, from plasmidCD4(181)-PE(392). The construction of CD4(181)-PE(392) is described byG. Winkler et al. ("CD4-Pseudomonas Exotoxin Hybrid Proteins: Modulationof Potency and Therapeutic Window Through Structural Design andCharacterization of Cell Internalization", AIDS Research and HumanRetroviruses, 7, pp. 393-401 (1991)). We inserted the ApaI-EcoRIfragment into pTat70(ApaI) digested with ApaI and EcoR1.

To construct plasmid pTat8PE (SEQ ID NO:11), we removed a 214-base pairNdeI-ApaI fragment from pTat70PE and replaced it with a double-strandedlinker having NdeI and ApaI cohesive termini, encoding tat residues 1-4and 67-70, and consisting of the following synthetic oligonucleotides:

    ______________________________________                                        TATGGAACCG GTCGTTTCTC TGTCGGGCC                                                                       (SEQ ID NO:12)                                        CGACAGAGAA ACGACCGGTT CCA                                                                             (SEQ ID NO:13).                                       ______________________________________                                    

Purification of TAT8-PE

Expression of the pTat8-PE construct yielded the PE ADP ribosylationdomain polypeptide fused to amino acids 1-4 and 67-70 of tat protein.The pTat8-PE expression product ("tat8-PE") served as the PE ADPribosylation domain moiety (and the unconjugated control) in chemicalcross-linking experiments described below. Codons for the 8 tat aminoacids were artifacts from a cloning procedure selected for convenience.The 8 tat amino acids fused to the PE ADP ribosylation domain had notransport activity (FIG. 13).

For purification of tat8-PE, we suspended 4.5 g of pTat8-PE-transformedE.coli in 20 ml of 50 mM Tris-HCl (pH 8.0), 2 mM EDTA. We lysed thecells in a French press and removed insoluble debris by centrifugationat 10,000 rpm for 1 hour, in an SA600 rotor. Most of the tat8-PE was inthe supernatant. We loaded the supernatant onto a 3 ml Q-Sepharose FastFlow (Pharmacia LKB, Piscataway, N.J.) ion exchange column. Afterloading the sample, we washed the column with 50 mM Tris-HCl (pH 8.0), 2mM EDTA. After washing the column, we carried out step gradient elution,using the same buffer with 100, 200 and 400 mM NaCl. The tat8-PE elutedwith 200 mM NaCl. Following the ion exchange chromatography, we furtherpurified the tat8-PE by gel filtration on a Superdex 75 FPLC column(Pharmacia LKB, Piscataway, N.J.). We equilibrated the gel filtrationcolumn with 50 mM HEPES (pH 7.5). We then loaded the sample and carriedout elution with the equilibration buffer at 0.34 ml/min. We collected1.5-minute fractions and stored the tat8-PE fractions at -70° C.

Crosslinking of TAT8-PE

Since the PE ADP ribosylation domain has no cysteine residues, we usedsulfo-SMCC (Pierce Chem. Co., Rockford, Ill. cat no. 22322 G) fortransport polypeptide-tat8-PE conjugation. We carried out theconjugation in a 2-step reaction procedure. In the first reaction step,we treated tat8-PE (3 mg/ml), in 50 mM HEPES (pH 7.5), with 10 mMsulfo-SMCC, at room temperature, for 40 minutes. (The sulfo-SMCC wasadded to the reaction as a 100 mM stock solution in 1M HEPES, pH 7.5.)We separated the tat8-PE-sulfo-SMCC from the unreacted sulfo-SMCC by gelfiltration on a P6DG column (Bio-Rad, Richmond, Calif.) equilibratedwith 25 mM HEPES (pH 6.0), 25 mM NaCl. In the second reaction step, weallowed the tat8-PE-sulfo-SMCC (1.5 mg/ml 100 mM HEPES (pH 7.5), 1 mMEDTA) to react with purified tat37-72 (600 μM final conc.) at roomtemperature, for 1 hour. To stop the cross-linking reaction, we addedcysteine. We analyzed the cross-linking reaction products by SDS-PAGE.About 90% of the tat8-PE became cross-linked to the tat37-72 transportpolypeptide under these conditions. Approximately half of the conjugatedproduct had one transport polypeptide moiety, and half had two transportpolypeptide moieties.

Cell-Free Assay for PE ADP Ribosylation

To verify that the PE ribosylation domain retained its biologicalactivity (i.e., destructive ribosome modification) following conjugationto transport polypeptides, we tested the effect of transportpolypeptide-PE ADP ribosylation conjugates on in vitro (i.e., cell-free)translation. For each in vitro translation experiment, we made up afresh translation cocktail and kept it on ice. The in vitro translationcocktail contained 200 μl rabbit reticulocyte lysate (Promega, Madison,Wis.), 2 μl 10 mM ZnCl₂ (optional), 4 μl of a mixture of the 20 proteinamino acids except methionine, and 20 μl ³⁵ S-methionine. To 9 μl oftranslation cocktail we added from 1 to 1000 ng of transportpolypeptide-PE conjugate (preferably in a volume of 1 μl) or control,and pre-incubated the mixture for 60 minutes at 30° C. We then added 0.5μl BMV RNA to each sample and incubated for an additional 60 minutes at30° C. We stored the samples at -70° C. after adding 5 μl of 50%glycerol per sample. We analyzed the in vitro translation reactionproducts by SDS-PAGE techniques. We loaded 2 μl of each translationreaction mixture (plus an appropriate volume of SDS-PAGE sample buffer)per lane on the SDS gels. After electrophoresis, we visualized the ³⁵S-containing in vitro translation products by fluorography.

Using the procedure described in the preceding paragraph, we found thatthe PE ADP ribosylation domain genetically fused to the tat1-70transport polypeptide had no biological activity, i.e., did not inhibitin vitro translation. In contrast, using the same procedure, we foundthat the PE ADP ribosylation domain chemically cross-linked to thetat37-72 transport polypeptide had retained full biological activity,i.e., inhibited in vitro translation as well as the non-conjugated PEADP ribosylation domain controls (FIG. 13).

Cytotoxicity Assay for PE ADP Ribosylation

In a further test involving the tat37-72-PE ADP ribosylation domainconjugate, we added it to cultured HeLa cells in the presence or absenceof 100 μM chloroquine. We then assayed cytotoxicity by measuring in vivoprotein synthesis, as indicated by trichloroacetic acid("TCA")-precipitable radioactivity in cell extracts.

We performed the cytotoxicity assay as follows. We disrupted HeLa celllayers, centrifuged the cells and resuspended them at a density of2.5×10⁴ /ml of medium. We used 0.5 ml of suspension/well when using 24well plates, or 0.25 ml of suspension/well when using 48 well plates. Weadded conjugates or unconjugated controls, dissolved in 100 μl of PBS,to the wells after allowing the cells to settle for at least 4 hours. Weincubated the cells in the presence of conjugates or controls for 60minutes, at 37° C., then added 0.5 ml of fresh medium to each cell, andincubated the cells for an additional 5-24 hours. Following thisincubation, we removed the medium from each well and washed the cellsonce with about 0.5 ml PBS. We then added 1 μCi of ³⁵ S-methionine(Amersham) per 100 μl per well in vivo cell labelling grade SJ.1015),and incubated the cells for 2 hours. After two hours, we removed theradioactive medium and washed the cells 3 times with cold 50% TCA andthen once with PBS. We added 100 μl of 0.5M NaOH to each well andallowed at least 45 minutes for cell lysis and protein dissolving totake place. We then added 50 μl 1M HCl to each well and transferred theentire contents of each well into scintillation fluid for liquidscintillation measurement of radioactivity.

In the absence of chloroquine, there was a clear dose-dependentinhibition of cellular protein synthesis in response to treatment withthe transport polypeptide-PE ADP ribosylation domain conjugate, but notin response to treatment with the unconjugated PE ADP ribosylationdomain. The results are summarized in FIG. 13. When conjugated totat37-72, the PE ADP ribosylation domain appeared to be transported 3 to10-fold more efficiently than when conjugated to tat1-72. We alsoconjugated transport polypeptides tat38-58GGC, tat37-58, tat47-58GGC andtatCGG-47-58 to the PE ADP ribosylation domain. All of these conjugatesresulted in cellular uptake of biologically active PE ADP ribosylationdomain (data not shown).

EXAMPLE 7 Ribonuclease Conjugates

Chemical Cross-Linking

We dissolved 7.2 mg of bovine pancreatic ribonuclease A, Type 12A (SigmaChem. Co., St. Louis, Mo., cat. no. R5500) in 200 μl PBS (pH 7.5). Tothe ribonuclease solution, we added 1.4 mg sulfo-SMCC (Pierce Chem. Co.,Rockford, Ill., cat. no. 22322H). After vortex mixing, we allowed thereaction to proceed at room temperature for 1 hour. We removed unreactedSMCC from the ribonuclease-SMCC by passing the reaction mixture over a 9ml P6DG column (Bio-Rad, Richmond, Calif.) and collecting 0.5 mlfractions. We identified the void volume peak fractions (containing theribonuclease-SMCC conjugate) by monitoring UV absorbance at 280 nm. Wedivided the pooled ribonuclease-SMCC-containing fractions into 5 equalaliquots. To each of 4 ribonuclease-SMCC aliquots, we added achemically-synthesized transport polypeptide corresponding to tatresidues: 37-72 ("37-72"); 38-58 plus GGC at the carboxy terminal("38-58GGC"); 37-58 ("CGG37-58"); or 47-58 plus CGG at the aminoterminal ("CGG47-58"). We allowed the transport polypeptide-ribonucleaseconjugation reactions to proceed for 2 hours at room temperature, andthen overnight at 4° C. We analyzed the reaction products by SDS-PAGE ona 10-20% gradient gel. The cross-linking efficiency was approximately60% for transport polypeptides tat38-58GGC, tat37-58 and tatCGG47-58,and 40% for Of the modified species, 72% contained one, and 25%contained 2 transport polypeptide substitutions.

Cellular Uptake of Tat37-72-Ribonuclease Conjugates

We maintained cells at 37° C. in a tissue culture incubator inDulbecco's Modified Eagle Medium supplemented with 10% donor calf serumand penicillium/streptomycin. For cellular uptake assays, we plated 10⁵cells in a 24-well plate and cultured them overnight. We washed thecells with Dulbecco's PBS and added the ribonuclease conjugate dissolvedin 300 μl of PBS containing 80 μM chloroquine, at concentrations of 0,10, 20, 40 and 80 μg/ml. After a 1.25 hour incubation at 37° C., weadded 750 μl of growth medium and further incubated the cell samplesovernight. After the overnight incubation, we washed the cells once withPBS and incubated them for 1 hour in Minimal Essential Medium withoutmethionine (Flow Labs) (250 μl/well) containing ³⁵ S methionine (1μCi/well). After the 1 hour incubation with radioactive methionine, weremoved the medium and washed the cells three times 5% TCA (1ml/well/wash). We then added 250 μl of 0.5M NaOH per well. After 1 hourat room temperature, we pipetted 200 μl of the contents of each wellinto a scintillation vial, added 100 μl of 1M HCl and 4 ml ofscintillation fluid. After thorough mixing of the contents of each vial,we measured radioactivity in each sample by liquid scintillationcounting.

The cellular uptake results are summarized in FIG. 3. Transportpolypeptide tat38-58GGC functioned as well as, or slightly better thantat37-72. Transport polypeptide tatCGG47-58 had reduced activity (datanot shown). We do not know whether this polypeptide had reduced uptakeactivity or whether the proximity of the basic region to theribonuclease interfered with enzyme activity.

We have used cation exchange chromatography (BioCAD perfusionchromatography system, PerSeptive Biosystems) to purify ribonucleaseconjugates having one or two transport polypeptide moieties.

EXAMPLE 8 Protein Kinase A Inhibitor Conjugates

Chemical Cross-Linking

We purchased the protein kinase A inhibitor ("PKAI") peptide (20 aminoacids) from Bachem California (Torrence, Calif.). For chemicalcross-linking of PKAI to transport polypeptides, we used eithersulfo-MBS (at 10 mM) or sulfo-SMPB (at 15 mM). Both of thesecross-linking reagents are heterobifunctional for thiol groups andprimary amine groups. Since PKAI lacks lysine and cysteine residues,both sulfo-MBS and sulfo-SMPB selectively target cross-linking to theamino terminus of PKAI. We reacted PKAI at a concentration of 2 mg/ml,in the presence of 50 mM HEPES (pH 7.5), 25 mM NaCl, at roomtemperature, for 50 minutes, with either cross-linking reagent. Thesulfo-MBS reaction mixture contained 10 mM sulfo-MBS and 20% DMF. Thesulfo-SMPB reaction mixture contained 15 mM sulfo-SMPB and 20%dimethylsulfoxide ("DMSO"). We purified the PKAI-cross-linker adducts byreverse phase HPLC, using a C₄ column. We eluted the samples from the C₄column in a 20-75% acetonitrile gradient containing 0.1% trifluoroaceticacid. We removed the acetonitrile and trifluoroacetic acid from theadducts by lyophilization and redissolved them in 25 mM HEPES (pH 6.0),25 mM NaCl. We added tat1-72 or tat37-72 and adjusted the pH of thereaction mixture to 7.5, by adding 1M HEPES (pH 7.5) to 100 mM. We thenallowed the cross-linking reaction to proceed at room temperature for 60minutes.

We regulated the extent of cross-linking by altering the transportpolypeptide:PKAI ratio. We analyzed the cross-linking reaction productsby SDS-PAGE. With tat37-72, a single new electrophoretic band formed inthe cross-linking reactions. This result was consistent with theaddition of a single tat37-72 molecule to a single PKAI molecule. Withtat1-72, six new products formed in the cross-linking reactions. Thisresult is consistent with the addition of multiple PKAI molecules pertat1-72 polypeptide, as a result of the multiple cysteine residues intat1-72. When we added PKAI to the cross-linking reaction in large molarexcess, we obtained only conjugates containing 5 or 6 PKAI moieties pertat1-72.

In Vitro Phosphorylation Assay for PKAI Activity

To test the sulfo-MBS-cross-linked conjugates for retention of PKAIbiological activity, we used an in vitro phosphorylation assay. In thisassay, histone V served as the substrate for phosphorylation by proteinkinase A in the presence or absence of PKAI (or a PKAI conjugate). Wethen used SDS-PAGE to monitor PKAI-dependent differences in the extentof phosphorylation. In each reaction, we incubated 5 units of thecatalytic subunit of protein kinase A Sigma) with varying amounts ofPKAI or PKAI conjugate, at 37° C., for 30 minutes. The assay reactionmixture contained 24 mM sodium acetate (pH 6.0), 25 mM MgCl₂, 100 mMDTT, 50 μCi of γ-³² P!ATP and 2 μg of histone V, in a total reactionvolume of 40 μl. Using this assay, we found that PKAI conjugated totat1-72 or tat37-72 inhibited phosphorylation as well as unconjugatedPKAI (data not shown).

Cellular Assay

To test for cellular uptake of PKAI and transport polypeptide-PKAIconjugates, we employed cultured cells containing a chloramphenicolacetyltransferase ("CAT") reporter gene under the control of acAMP-responsive expression control sequence. We thus quantified proteinkinase A activity indirectly, by measuring CAT activity. This assay hasbeen described in detail by J. R. Grove et al. ("Probind cAMP-RelatedGene Expression with a Recombinant Protein Kinase Inhibitor", MolecularAspects of Cellular Regulation, Vol. 6, P. Cohen and J. G. Folkes, eds.,Elsevier Scientific, Amsterdam, pp. 173-95 (1991)).

Using this assay, we found no activity by PKAI or any of the transportpolypeptide-PKAI conjugates. This result suggested to us that the PKAImoiety might be undergoing rapid degradation upon entry into the cells.

Cross-Linking of PKAI to Tat37-72-β-Galactosidase

We had previously found cellular uptake of tat37-72-β-galactosidase tobe chloroquine-independent (Example 2, above). Therefore, wecross-linked PKAI to tat37-72-β-galactosidase for possible protection ofPKAI against rapid degradation.

We treated β-galactosidase with 20 mM DTT (a reducing agent) at roomtemperature for 30 minutes and then removed the DTT by gel filtration ona G50 column in MES buffer (pH 5). We allowed the reducedβ-galactosidase to react with SMPB-activated PKAI (above), at pH 6.5,for 60 minutes. To block residual free sulfhydryl groups, we addedN-ethylmaleimide or iodoacetamide. SDS-PAGE analysis showed that atleast 95% of the β-galactosidase had been conjugated. About 90% of theconjugated beta-galactosidase product contained one PKAI moiety persubunit, and about 10% contained 2 PKAI moieties. We treated thePKAI-β-galactosidase conjugate with a 10-fold molar excess ofsulfo-SMCC. We then reacted the PKAI-β-galactosidase-SMCC with tat1-72.According to SDS-PAGE analysis, the PKAI-β-galactosidase:tat1-72 ratioappeared to be 1:0.5. We have produced about 100 μg of the finalproduct. Because of precipitation problems, the concentration of thefinal product in solution has been limited to 100 μg/ml.

EXAMPLE 9 E2 Repressor Conjugates

To test cellular uptake and E2 repressor activity of transportpolypeptide-E2 repressor conjugates, we simultaneously transfected anE2-dependent reporter plasmid and an E2 expression plasmid intoSV40-transformed African green monkey kidney ("COS7") cells. Then weexposed the transfected cells to transport polypeptide-E2 repressorconjugates (made by genetic fusion or chemical cross-linking) or toappropriate controls. The repression assay, described below, wasessentially as described in Barsoum et al. (supra).

Repression Assay Cells

We obtained the COS7 cells from the American Type Culture Collection,Rockville, Md. (ATCC No. CRL 1651). We propagated the COS7 cells inDulbecco's modified Eagle's medium (GIBCO, Grand Island, N.Y.) with 10%fetal bovine serum (JRH Biosciences, Lenexa, Kans.) and 4 mM glutamine("growth medium"). Cell incubation conditions were 5.5% CO₂ at 37° C.

Repression Assay Plasmids

Our E2-dependent reporter plasmid, pXB332hGH, contained a human growthhormone reporter gene driven by a truncated SV40 early promoter having 3upstream E2 binding sites. We constructed the hGH reporter plasmid,pXB332hGH, as described in Barsoum et al. (supra).

For expression of a full-length HPV E2 gene, we constructed plasmidpAHE2 (FIG. 15). Plasmid pAHE2 contains the E2 gene from HPV strain 16,operatively linked to the adenovirus major late promoter augmented bythe SV40 enhancer, upstream of the promoter. We isolated the HPV E2 genefrom plasmid pHPV16 (the full-length HPV16 genome cloned into pBR322),described in M. Durst et al., "A Papillomavirus DNA from CervicalCarcinoma and Its Prevalence in Cancer Biopsy Samples from DifferentGeographic Regions", Proc. Natl. Acad. Sci. USA, 80, pp. 3812-15 (1983),as a Tth111I-AseI fragment. Tth111I cleaves at nucleotide 2711, and AseIcleaves at nucleotide 3929 in the HPV16 genome. We blunted the ends ofthe Tth111I-AseI fragment in a DNA polymerase I Klenow reaction, andligated BamHI linkers (New England Biolabs, cat. no. 1021). We insertedthis linker-bearing fragment into BamHI-cleaved plasmid pBG331, tocreate plasmid pAHE2.

Plasmid pBG331 is the same as pBG312 (R. L. Cate et al., "Isolation ofthe Bovine and Human Genes for Mullerian Inhibiting Substance andExpression of the Human Gene in Animal Cells", Cell, 45, pp. 685-98(1986)) except that it lacks the BamHI site downstream of the SV40polyadenylation signal, making the BamHI site between the promoter andthe SV40 intron unique. We removed the unwanted BamHI site by partialBamHI digestion of pBG312, gel purification of the linearized plasmid,blunt end formation by DNA polymerase I Klenow treatment, self-ligationand screening for plasmids with the desired deletion of the BamHI site.

Bacterial Production of E2 Repressor Proteins

One of our E2 repressor proteins, E2.123, consisted of thecarboxy-terminal 121 amino acids of HPV16 E2 with MetVal added at theamino terminus. We also used a variant of E2.123, called E2.123CCSS.E2.123 has cysteine residues at HPV16 E2 amino acid positions 251, 281,300 and 309. In E2.123CCSS, the cysteine residues at positions 300 and309 were changed to serine, and the lysine residue at position 299 waschanged to arginine. We replaced the cysteine residues at positions 300and 309, so that cysteine-dependent chemical cross-linking could takeplace in the amino terminal portion of the E2 repressor, but not in theE2 minimal DNA binding/dimerization domain. We considered crosslinks inthe minimal DNA binding domain likely to interfere with the repressor'sbiological activity.

For construction of plasmid pET8c-123 (FIG. 16; SEQ ID NO:14), weproduced the necessary DNA fragment by standard polymerase chainreaction ("PCR") techniques, with plasmid pHPV16 as the template. (For ageneral discussion of PCR techniques, see Chapter 14 of Sambrook et al.,supra. Automated PCR equipment and chemicals are commerciallyavailable.) The nucleotide sequence of EA52, the PCR oligonucleotideprimer for the 5' end of the 374 base pair E2-123 fragment, is set forthin the Sequence Listing under SEQ ID NO:15. The nucleotide sequence ofEA54, the PCR oligonucleotide primer used for the 3' end of the E2-123fragment is set forth in the Sequence Listing under SEQ ID NO:16. Wedigested the PCR products with NcoI and BamHI and cloned the resultingfragment into NcoI/BamHI-digested expression plasmid pET8c (Studier etal., supra), to create plasmid pET8c-123.

By using the same procedure with a different 5' oligonucleotide PCRprimer, we obtained a 260 base pair fragment ("E2-85") containing amethionine codon and an alanine codon immediately followed by codons forthe carboxy-terminal 83 amino acids of HPV16 E2. The nucleotide sequenceof EA57, the PCR 5' primer for producing E2-85, is set forth in theSequence Listing under SEQ ID NO:34.

To construct plasmid pET8c-123CCSS (FIG. 17; SEQ ID NO:17), forbacterial production of E2.123CCSS, we synthesized an 882 bp PstI-EagIDNA fragment by PCR techniques. The PCR template was pET8c-123. One ofthe PCR primers, called 374.140, encoded all three amino acid changes:CGACACTGCA GTATACAATG TAGAATGCTT TTTAAATCTA TATCTTAAAG ATCTTAAAG (SEQ IDNO:18). The other PCR primer, 374.18, had the following sequence:GCGTCGGCCG CCATGCCGGC GATAAT (SEQ ID NO:19). We digested the PCRreaction products with PstI plus EagI and isolated the 882 bp fragmentby standard methods. The final step was production of pET8c-123CCSS in a3-piece ligation joining a 3424 bp EcoRI-EagI fragment from pET8c-123with the 882 bp PCR fragment and a 674-bp PstI-EcoRI pET8c-123 fragment,as shown in FIG. 17. We verified the construction by DNA sequenceanalysis. For production of E2.123 and E2.123CCSS proteins, we expressedplasmids pET8c-123 and pET8c-123CCSS in E.coli strain BL21(DE3)pLysS, asdescribed by Studier (supra).

Purification of E2 Repressor Proteins

We thawed 3.6 grams of frozen, pET8c-123-transformed E.coli cells andsuspended them in 35 ml of 25 mM Tris-HCl (pH 7.5), 0.5 mM EDTA, 2.5 mMDTT, plus protease inhibitors (1 mM PMSF, 3 mM benzamidine, 50 μg/mlpepstatin A, 10 μg/ml aprotinin). We lysed the cells by two passagesthrough a French press at 10,000 psi. We centrifuged the lysate at12,000 rpm, in an SA600 rotor, for 1 hour. The E2.123 protein was in thesupernatant. To the supernatant, we added MES buffer (pH 6) up to 25 mM,MES buffer (pH 5) up to 10 mM, and NaCl up to 125 mM. We then appliedthe supernatant to a 2 ml S Sepharose Fast Flow column at 6 ml/hr. Afterloading, we washed the column with 50 mM Tris-HCl (pH 7.5), 1 mM DTT. Wethen carried out step gradient elution (2 ml/step) with 200, 300, 400,500, 700 and 1000 mM NaCl in 50 mM Tris-HCl (pH 7.5), 1 mM DTT. TheE2.123 repressor protein eluted in the 500 and 700 mM NaCl fractions.SDS-PAGE analysis indicated the E2.123 repressor purity exceeded 95%.

We thawed 3.0 grams of frozen, pET8c-123CCSS-transformed E.coli andsuspended the cells in 30 ml of the same buffer used forpET8c-123-transformed cells (above). Lysis, removal of insolublecellular debris and addition of MES buffer and NaCl was also asdescribed for purification of E2-123. The purification procedure forE2.123CCSS diverged after addition of the MES buffer and NaCl, because aprecipitate formed, with E2.123CCSS, at that point in the procedure. Weremoved the precipitate by centrifugation, and found that it and thesupernatant both contained substantial E2 repressor activity. Therefore,we subjected both to purification steps. We applied the supernatant to a2 ml S Sepharose Fast Flow column (Pharmacia LKB, Piscataway, N.J.) at 6ml/hr. After loading, we washed the column with 50 mM Tris-HCl (pH 7.5),1 mM DTT. After washing the column, we carried out step gradient elution(2 ml/step), using 300, 400, 500, 700 and 1000 mM NaCl in 50 mM Tris-HCl(pH 7.5), 1 mM DTT. The E2.123CCSS protein eluted with 700 mM NaCl.SDS-PAGE analysis indicated its purity to exceed 95%. We dissolved theE2.123CCSS precipitate in 7.5 ml of 25 mM Tris-HCl (pH 7.5), 125 mMNaCl, 1 mM DTT and 0.5 mM EDTA. We loaded the dissolved material onto a2 ml S Sepharose Fast Flow column and washed the column as described forE2.123 and non-precipitated E2.123CCSS. We carried out step gradientelution (2 ml/step), using 300, 500, 700 and 1000 mM NaCl. The E2repressor eluted in the 500-700 mM NaCl fractions. SDS-PAGE analysisindicated its purity to exceed 98%. Immediately following purificationof the E2.123 and E2.123CCSS proteins, we added glycerol to a finalconcentration of 15% (v/v), and stored flash-frozen (liquid N₂) aliquotsat -70° C. We quantified the purified E2 repressor proteins by UVabsorbance at 280 nm, using an extinction coefficient of 1.8 at 1 mg/ml.

Chemical Cross-Linking

We performed chemical synthesis of the transport polypeptide consistingof tat amino acids 37-72, as described in Example 1. We dissolved thepolypeptide (5 mg/ml) in 10 mM MES buffer (pH 5.0), 50 mM NaCl, 0.5 mMEDTA, (extinction coefficient of 0.2 at 1 ml/ml). To the transportpolypeptide solution, we added a bismaleimidohexane ("BMH") (PierceChemical Co., Rockford, Ill., cat. no. 22319G) stock solution (6.25mg/ml DMF) to a final concentration of 1.25 mg/ml, and a pH 7.5 HEPESbuffer stock solution (1M) to a final concentration of 100 mM. Weallowed the BMH to react with the protein for 30 minutes at roomtemperature. We then separated the protein-BMH from unreacted BMH by gelfiltration on a G-10 column equilibrated in 10 mM MES (pH 5), 50 mMNaCl, 0.5 mM EDTA. We stored aliquots of the transport polypeptide-BMHconjugate at -70° C.

For cross-linking of the transport polypeptide-BMH conjugate to the E2repressor, we removed the E2 repressor protein from its storage buffer.We diluted the E2 repressor protein with three volumes of 25 mM MES (pH6.0), 0.5 mM EDTA and batch-loaded it onto S Sepharose Fast Flow(Pharmacia LKB, Piscataway, N.J.) at 5 mg protein per ml resin. Afterpouring the slurry of protein-loaded resin into a column, we washed thecolumn with 25 mM MES (pH 6.0), 0.5 mM EDTA, 250 mM NaCl. We then elutedthe bound E2 repressor protein from the column with the same buffercontaining 800 mM NaCl. We diluted the E2 repressor-containing eluate to1 mg/ml with 25 mM MES (pH 6.0), 0.5 mM EDTA. From trial cross-linkingstudies performed with each batch of E2 repressor protein andBMH-activated transport polypeptide, we determined that treating 1 mg ofE2 repressor protein with 0.6 mg of BMH-activated transport polypeptideyields the desired incorporation of 1 transport molecule per E2repressor homodimer. Typically, we mixed 2 ml of E2 repressor (1 mg/ml)with 300 μl of tat37-72-BMH (4 mg/ml) and 200 μl of 1M HEPES (pH 7.5).We allowed the cross-linking reaction to proceed for 30 minutes at roomtemperature. We terminated the cross-linking reaction by adding2-mercaptoethanol to a final concentration of 14 mM. We determined theextent of cross-linking by SDS-PAGE analysis. We stored aliquots of thetat37-72-E2 repressor conjugate at -70° C. We employed identicalprocedures to chemically cross-link the tat37-72 transport polypeptideto the HPVE2 123 repressor protein and the HPVE2 CCSS repressor protein.

Cellular Uptake of E2 Repressor Conjugates

For our E2 repression assays, we used transient expression of plasmidstransfected into COS7 cells. Our E2 repression assay procedure wassimilar to that described in Barsoum et al. (supra). We transfected4×10⁶ COS7 cells (about 50% confluent at the time of harvest) byelectroporation, in two separate transfections ("EP1" and "EP2"). Intransfection EP1, we used 20 μg pXB332hGH (reporter plasmid) plus 380 μgsonicated salmon sperm carrier DNA (Pharmacia LKB, Piscataway, N.J.). Intransfection EP2, we used 20 μg pXB332hGH plus 30 μg pAHE2 (E2transactivator) and 350 μg salmon sperm carrier DNA. We carried outelectroporations with a Bio-Rad Gene Pulser, at 270 volts, 960 μFD, witha pulse time of about 11 msec. Following the electroporations, we seededthe cells in 6-well dishes, at 2×10⁵ cells per well. Five hours afterthe electroporations, we aspirated the growth medium, rinsed the cellswith growth medium and added 1.5 ml of fresh growth medium to each well.At this time, we added chloroquine ("CQ") to a final concentration of 80μM (or a blank solution to controls). Then we added tat37-72cross-linked E2.123 ("TxHE2") or tat37-72 cross-linked to E2.123CCSS("TxHE2CCSS"). The final concentration of these transportpolypeptide-cargo conjugates was 6, 20 or 60 μg/ml of cell growth medium(Table I).

                  TABLE I                                                         ______________________________________                                        Identification of Samples                                                     well     CO (μM)      protein (μg/ml)                                   ______________________________________                                        EP1.1     0               0                                                   EP1.2    80               0                                                   EP2.1     0               0                                                   EP2.2     0               6 TxHE2                                             EP2.3     0              20 TxHE2                                             EP2.4     0              60 TxHE2                                             EP2.5     0               6 TxHE2CCSS                                         EP2.6     0              20 TxHE2CCSS                                         EP2.7     0              60 TxHE2CCSS                                         EP2.8    80               0                                                   EP2.9    80               6 TxHE2                                             EP2.10   80              20 TxHE2                                             EP2.11   80              60 TxHE2                                             EP2.12   80               6 TxHE2CCSS                                         EP2.13   80              20 TxHE2CCSS                                         EP2.14   80              60 TxHE2CCSS                                         ______________________________________                                    

After an 18-hour incubation, we removed the medium, rinsed the cellswith fresh medium, and added 1.5 ml of fresh medium containing the sameconcentrations of chloroquine and transport polypeptide-cargo conjugatesas in the preceding 18-hour incubation. This medium change was to removeany hGH that may have been present before the repressor entered thecells. Twenty-four hours after the medium change, we harvested the cellsand performed cell counts to check for viability. We then assayed forhGH on undiluted samples of growth medium according to the method ofSeldon, described in Protocols in Molecular Biology, Green PublishingAssociates, New York, pp. 9.7.1-9.7.2 (1987), using the Allegro HumanGrowth Hormone transient gene expression system kit (Nichols Institute,San Juan Capistrano, Calif.). We subtracted the assay background (i.e.,assay components with non-conditioned medium added) from the hGH cpm,for all samples. We performed separate percentage repressioncalculations for a given protein treatment, according to whetherchloroquine was present ("(+)CQ") or absent ("(-)CQ") in the proteinuptake test. We calculated percentage repression according to thefollowing formula: ##EQU1## where: BKG=hGH cpm in the transfections ofreporter alone (e.g., EP1.1 for (-)CQ and EP1.2 for (+)CQ);

ACT=hGH cpm in the transfection of reporter plus transactivator, but towhich no repressor conjugate was added (e.g., EP2.1 for (-)CQ and EP2.8for (+)CQ);

REP=hGH cpm in the transfection of reporter plus transactivator, towhich a repressor conjugate was added (e.g., EP2.2-2.7 for (-)CQ andEP2.9-2.14 for (+)CQ).

Data from a representative E2 repression assay are shown in Table II.Table I identifies the various samples represented in Table II. FIG. 18graphically depicts the results presented in Table II.

                  TABLE II                                                        ______________________________________                                        E2 Repression Assay                                                                           cpm -                                                         sample hGH cpm  assay bkgd  cpm - BKG                                                                             % repression                              ______________________________________                                        EP1.1  3958     3808        --      --                                        EP1.2  5401     5251        --      --                                        EP2.1  15,161   15,011      11,203  --                                        EP2.2  12,821   12,671      8863    20.9                                      EP2.3  10,268   10,118      6310    43.7                                      EP2.4  8496     8346        4538    59.5                                      EP2.5  11,934   11,784      7976    28.8                                      EP2.6  9240     9090        5282    52.9                                      EP2.7  7926     7776        3968    64.6                                      EP2.8  15,120   14,970      9719    --                                        EP2.9  12,729   12,579      7328    24.6                                      EP2.10 9590     9440        4189    56.9                                      EP2.11 8440     8290        3039    68.7                                      EP2.12 11,845   11,695      6444    33.7                                      EP2.13 8175     8025        2774    71.5                                      EP2.14 6697     6547        1296    86.7                                      ______________________________________                                    

Transport polypeptide tat37-72 cross-linked to either E2 repressor(E2.123 or E2.123CCSS) resulted in a dose-dependent inhibition ofE2-dependent gene expression in the cultured mammalian cells (Table II;FIG. 18). We have repeated this experiment four times, with similarresults. The effect was E2-specific, in that other tat37-72 conjugateshad no effect on E2 induction of pXB332hGH (data not shown). Also, thetat37-72xHE2 conjugates had no effect on the hGH expression level of areporter in which the expression of the hGH gene was driven by aconstitutive promoter which did not respond to E2. The E2 repressor withthe CCSS mutation repressed to a greater degree than the repressor withthe wild-type amino acid sequence. This was as expected, becausecross-linking of the transport polypeptide to either of the last twocysteines in the wild-type repressor would likely reduce or eliminaterepressor activity. Chloroquine was not required for the repressionactivity. However, chloroquine did enhance repression in all of thetests. These results are summarized in Table II and FIG. 18.

EXAMPLE 10 TATΔCYS Conjugates

Production of TatΔcys

For bacterial production of a transport polypeptide consisting of tatamino acids 1-21 fused directly to tat amino acids 38-72, we constructedexpression plasmid pTATΔcys (FIG. 19; SEQ ID NO:20). To constructplasmid pTATΔcys, we used conventional PCR techniques, with plasmidpTAT72 as the PCR template. One of the oligonucleotide primers used forthe PCR was 374.18 (SEQ ID NO:19), which covers the EagI site upstreamof the tat coding sequence. (We also used oligonucleotide 374.18 in theconstruction of plasmid pET8c-123CCSS. See Example 9.) The otheroligonucleotide primer for the PCR, 374.28, covers the EagI site withinthe tat coding sequence and has a deletion of the tat DNA sequenceencoding amino acids 22-37. The nucleotide sequence of 374.28 is:TTTACGGCCG TAAGAGATAC CTAGGGCTTT GGTGATGAAC GCGGT (SEQ ID NO:21). Wedigested the PCR products with EagI and isolated the resulting 762-basepair fragment. We inserted that EagI fragment into the 4057 base pairvector produced by EagI cleavage of pTAT72. We verified the constructionby DNA sequence analysis and expressed the tatΔcys polypeptide by themethod of Studier et al. (supra). SDS-PAGE analysis showed the tatΔcyspolypeptide to have the correct size.

For purification of tatΔcys protein, we thawed 4.5 grams ofpTATΔcys-transformed E.coli cells, resuspended the cells in 35 ml of 20mM MES (pH 6.2), 0.5 mM EDTA. We lysed the cells by two passes through aFrench press, at 10,000 psi. We removed insoluble debris bycentrifugation at 10,000 rpm in an SA600 rotor, for 1 hour. We appliedthe supernatant to a 5 ml S Sepharose Fast Flow column at 15 ml/hr. Wewashed the column with 50 mM Tris-HCl (pH 7.5), 0.3 mM DTT. We thencarried out step gradient elution (2 ml/step) with the same buffercontaining 300, 400, 500, 700 and 950 mM NaCl. The tatΔcys proteineluted in the 950 mM NaCl fraction.

We conjugated a tatΔcys transport polypeptide to rhodamineisothiocyanate and tested it by assaying directly for cellular uptake.The results were positive (similar to results in related experimentswith tat1-72).

TATΔcys-249 Genetic Fusion

For bacterial expression of the tatΔcys transport polypeptidegenetically fused to the amino terminus of the native E2 repressorprotein (i.e., the carboxy-terminal 249 amino acids of BPV-1 E2), weconstructed plasmid pTATΔcys-249 as follows. We constructed plasmidpFTE501 (FIG. 20) from plasmids pTAT72 (Frankel and Pabo, supra andExample I) and pXB314 (Barsoum et al., supra). From plasmid pXB314, weisolated the NcoI-SpeI DNA fragment encoding the 249 amino acid BPV-1 E2repressor. (NcoI cleaves at nucleotide 296, and SpeI cleaves atnucleotide 1118 of pXB314.) We blunted the ends of this fragment by DNApolymerase I Klenow treatment and added a commercially available BglIIlinker (New England Biolabs, cat. no. 1090). We inserted thislinker-bearing fragment into BamHI-cleaved (complete digestion) plasmidpTAT72. In pTAT72, there is a BamHI cleavage site within the tat codingregion, near its 3' end, and a second BamHI cleavage site slightlydownstream of the tat gene. The BglII linker joined the tat and E2coding sequences in frame to encode a fusion of the first 62 amino acidsof tat protein followed by a serine residue and the last 249 amino acidsof BPV-1 E2 protein. We designated this bacterial expression plasmidpFTE501 (FIG. 20). To construct plasmid pTATΔcys-249 (FIG. 21; SEQ IDNO:22), we inserted the 762 base pair EagI fragment from plasmid pTATcys, which includes the portion of tat containing the cysteine deletion,into the 4812 base pair EagI fragment of plasmid pFTE501.

Purification of tatΔcys-249

We thawed 5 g of E.coli expressing tatΔcys-249 and suspended the cellsin 40 ml of 25 mM Tris HCl (pH 7.5), 25 mM NaCl, 0.5 mM EDTA, 5 mM DTT,plus protease inhibitors (1.25 mM PMSF, 3 mM Benzamidine, 50 μg/mlpepstatin A, 50 μg/ml aprotinin, 4 μg/ml E64). We lysed the cells by twopassages through a French pressure cell at 10,000 psi. We removedinsoluble debris from the lysate by centrifugation at 12,000 rpm in anSA600 rotor, for 1 hour. We purified the tatΔcys-249 from the solublefraction. The supernatant was loaded onto a 2 ml S Sepharose Fast Flowcolumn (Pharmacia LKB, Piscataway, N.J.) at a flow rate of 6 ml/h. Thecolumn was washed with 25 mM Tris HCl pH (7.5), 25 mM NaCl, 0.5 mM EDTA,1 mM DTT and treated with sequential salt steps in the same buffercontaining 100, 200, 300, 400, 500, 600, and 800 mM NaCl. We recoveredthe TatΔcys-249 in the 600-800 mM salt fractions. We pooled the peakfractions, added glycerol to 15%, and stored aliquots at -70° C.

Immunofluorescence Assay

To analyze cellular uptake of the tatΔcys-E2 repressor fusion protein,we used indirect immunofluorescence techniques. We seeded HeLa cellsonto cover slips in 6-well tissue culture dishes, to 50% confluence.After an overnight incubation, we added the tatΔcys-E2 repressor fusionprotein (1 μg/ml final concentration) and chloroquine (0.1 mM finalconcentration). After six hours, we removed the fusionprotein/chloroquine-containing growth medium and washed the cells twicewith PBS. We fixed the washed cells in 3.5% formaldehyde at roomtemperature. We permeabilized the fixed cells with 0.2% Triton X-100/2%bovine serum albumin ("BSA") in PBS containing 1 mM MgCl₂ /0.1 mM CaCl₂("PBS+") for 5 minutes at room temperature. To block the permeabilizedcells, we treated them with PBS containing 2% BSA, for 1 hour at 4° C.

We incubated the cover slips with 20 μl of a primary antibody solutionin each well, at a 1:100 dilution in PBS+ containing 2% BSA, for 1 hourat 4° C. The primary antibody was either a rabbit polyclonal antibody tothe BPV-1 E2 repressor (generated by injecting the purifiedcarboxy-terminal 85 amino acids of E2), or a rabbit polyclonal antibodyto tat (generated by injecting the purified amino-terminal 72 aminoacids of tat protein). We added a secondary antibody at a 1:100 dilutionin 0.2% Tween-20/2% BSA in PBS+ for 30 minutes at 4° C.

The secondary antibody was a rhodamine-conjugated goat anti-rabbit IgG(Cappel no. 2212-0081). Following incubation of the cells with thesecondary antibody, we washed the cells with 0.2% Tween 20/2% BSA inPBS+ and mounted the cover slips in 90% glycerol, 25 mM sodium phosphate(pH 7.2), 150 mM NaCl. We examined the cells with a fluorescentmicroscope having a rhodamine filter.

Cellular Uptake of TatΔCys Fusions

We observed significant cellular uptake of the tatΔcys-E2 repressorfusion protein, using either the tat antibody or the E2 antibody. Incontrol cells exposed to the unconjugated tat protein, we observedintracellular fluorescence using the tat antibody, but not the E2antibody. In control cells exposed to a mixture of the unconjugated E2repressor and tat protein or tatΔcys, we observed fluorescence using thetat antibody, but not the E2 antibody. This verified that tat mediatesE2 repressor uptake only when linked to the tat protein. As withunconjugated tat protein, we observed the tatΔcys-E2 repressor fusionprotein throughout the cells, but it was concentrated in intracellularvesicles. These results show that a tat-derived polypeptide completelylacking cysteine residues can carry a heterologous protein (i.e.,transport polypeptide-cargo protein genetic fusion) into animal cells.

In a procedure similar to that described above, we produced a geneticfusion of tatΔcys to the C-terminal 123 amino acids of HPV E2. Whenadded to the growth medium, this fusion polypeptide exhibited repressionof E2-dependent gene expression in COS7 cells (data not shown).

EXAMPLE 11 Antisense Oligodeoxynucleotide Conjugates

Using an automated DNA/RNA synthesizer (Applied Biosystems model 394),we synthesized DNA phosphorothionate analogs (4-18 nucleotides inlength), with each containing a free amino group at the 5' end. Theamine group was incorporated into the oligonucleotides usingcommercially modified nucleotides (aminolink 2, Applied Biosystems). Theoligonucleotides corresponded to sense and antisense strands fromregions of human growth hormone and CAT messenger RNA.

For each cross-linking reaction, we dissolved 200 μg of anoligonucleotide in 100 μl of 25 mM sodium phosphate buffer (pH 7.0). Wethen added 10 μl of a 50 mM stock solution of sulfo-SMCC and allowed thereaction to proceed at room temperature for 1 hour. We removed unreactedsulfo-SMCC by gel filtration of the reaction mixture on a P6DG column(Bio-Rad) in 25 mM HEPES (pH 6.0). We dried theoligonucleotide-sulfo-SMCC adduct under a vacuum. Recovery of theoligonucleotides in this procedure ranged from 58 to 95%. For reactionwith a transport polypeptide, we redissolved eacholigonucleotide-sulfo-SMCC adduct in 50 μl of 0.5 mM EDTA, transferredthe solution to a test tube containing 50 μg of lyophilized transportpolypeptide, and allowed the reaction to proceed at room temperature for2 hours. We analyzed the reaction products by SDS-PAGE.

EXAMPLE 12 Antibody Conjugates

Anti-Tubulin Conjugate 1

We obtained commercial mouse IgG1 mAb anti-tubulin (Amersham) andpurified it from ascites by conventional methods, using protein A. Welabelled the purified antibody with rhodamine isothiocyanate, at 1.2moles rhodamine/mole Ab. When we exposed fixed, permeabilized HeLa cellsto the labelled antibody, microscopic examination revealed brightlystained microtubules. Although the rhodamine labelling was sufficient,we enhanced the antibody signal with anti-mouse FITC.

In a procedure essentially as described in Example 2, (above) we allowed250 μg of the antibody to react with a 10:1 molar excess of sulfo-SMCC.We then added 48 μg of (³⁵ S-labelled) tat1-72. The molar ratio oftat1-71:Ab was 2.7:1. According to incorporation of radioactivity, thetat1:72 was cross-linked to the antibody in a ratio of 0.6:1.

For analysis of uptake of the tat1-72-Ab conjugate, we added theconjugate to medium (10 μg/ml) bathing cells grown on coverslips. Weobserved a punctate pattern of fluorescence in the cell. The punctatepattern indicated vesicular location of the conjugate, and was thereforeinconclusive as to cytoplasmic delivery.

To demonstrate immunoreactivity of the conjugated antibody, we testedits ability to bind tubulin. We coupled purified tubulin to cyanogenbromide-activated Sepharose 4B (Sigma Chem. Co., St. Louis, Mo.). Weapplied a samples of the radioactive conjugate to the tubulin column(and to a Sepharose 4B control column) and measured the amount of boundconjugate. More radioactivity bound to the affinity matrix than to thecontrol column, indicating tubulin binding activity.

Anti-Tubulin Conjugate 2

In a separate cross-linking experiment, we obtained an anti-tubulin ratmonoclonal antibody IgG2a (Serotec), and purified it from ascites byconventional procedures, using protein G. We eluted the antibody withCaps buffer (pH 10). The purified antibody was positive in atubulin-binding assay. We allowed tat1-72 to react with rhodamineisothiocyanate at a molar ratio of 1:1. The reaction product exhibitedan A₅₅₅ /A₂₈₀ ratio of 0.63, which indicated a substitution ofapproximately 0.75 mole of dye per mole of tat1-72. Upon separation ofthe unreacted dye from the tat1-72-rhodamine, by G-25 gel filtration(Pharmacia LKB, Piscataway, N.J.), we recovered only 52 μg out of 150 μgof tat1-72 used in the reaction.

We saved an aliquot of the tat1-72-rhodamine for use (as a control) incellular uptake experiments, and added the rest to 0.4 mg of antibodythat had reacted with SMCC (20:1). The reaction mixture contained atat1-72:Ab ratio of approximately 1:1, rather than the intended 5:1. (Ina subsequent experiment, the 5:1 ratio turned out to be unsatisfactory,yielding a precipitate.) We allowed the cross-linking reaction toproceed overnight at 4° C. We then added a molar excess of cysteine toblock the remaining maleimide groups and thus stop the cross-linkingreaction. We centrifuged the reaction mixtures to remove any precipitatepresent.

We carried out electrophoresis using a 4-20% polyacrylamide gradient gelto analyze the supernatant under reducing and non-reducing conditions.We also analyzed the pellets by this procedure. In supernatants fromantibody-tat1-72 (without rhodamine) conjugation experiments, weobserved very little material on the 4-20% gel. However, in supernatantsfrom the antibody-tat1-72-rhodamine conjugation experiments, we observedrelatively heavy bands above the antibody, for the reduced sample. Theantibody appeared to be conjugated to the tat1-72 in a ratio ofapproximately 1:1.

In cellular uptake experiments carried out with conjugate 2 (procedureas described above for conjugate 1), we obtained results similar tothose obtained with conjugate 1. When visualizing the conjugate byrhodamine fluorescence or by fluorescein associated with a secondantibody, we observed the conjugate in vesicles.

EXAMPLE 13 Additional Tat-E2 Conjugates

Chemically Cross-Linked Tat-E2 Conjugates

We chemically cross-linked transport polypeptide tat37-72 to fourdifferent repressor forms of E2. The four E2 repressor moieties used inthese experiments were the carboxy-terminal 103 residues (i.e., 308-410)of BPV-1 ("E2.103"); the carboxy-terminal 249 residues (i.e., 162-410)of BPV-1 ("E2.249"); the carboxy-terminal 121 residues (i.e., 245-365)of HPV-16 ("HE2"); and the carboxy-terminal 121 residues of HPV-16, inwhich the cysteine residues at positions 300 and 309 were changed toserine, and the lysine residue at position 299 was changed to arginine("HE2CCSS"). The recombinant production and purification of HE2 andHE2CCSS, followed by chemical cross-linking of HE2 and HE2CCSS totat37-72, to form TxHE2 and TxHE2CCSS, respectively, are described inExample 9 (above). For the chemical cross-linking of E2.103 and E2.249to tat37-72 (to yield the conjugates designated TxE2.103 and TxE2.249),we employed the same method used to make TxHE2 and TxHE2CCSS (Example 9,supra).

We expressed the protein E2.103 in E.coli from plasmid pET-E2.103. Weobtained pET-E2.103 by a PCR cloning procedure analogous to that used toproduce pET8c-123, described in Example 9 (above) and FIG. 16. As in theconstruction of pET8c-123, we ligated a PCR-produced NcoI-BamHI E2fragment into NcoI-BamHI-cleaved pET8c. Our PCR template for the E2fragment was plasmid pCO-E2 (Hawley-Nelson et al., EMBO J., vol 7, pp.525-31 (1988); U.S. Pat. No. 5,219,990). The oligonucleotide primersused to produce the E2 fragment from pCO-E2 were EA21 (SEQ ID NO:36) andEA22 (SEQ ID NO:37). Primer EA21 introduced an NcoI site that added amethionine codon followed by an alanine codon 5' adjacent to the codingregion for the carboxy-terminal 101 residues of BPV-1 E2.

We expressed the protein E2.249 in E.coli from plasmid pET8c-249. Weconstructed pET8c-249 by inserting the 1362 bp NcoI-BamHI fragment ofplasmid pXB314 (FIG. 20) into NcoI-BamHI-cleaved pET8c (FIG. 16).

TATΔcys-BPV E2 Genetic Fusions

In addition to TATΔcys-249, we tested several other TATΔcys-BPV-1 E2repressor fusions. Plasmid pTATΔcys-105 encoded tat residues 1-21 and38-67, followed by the carboxy-terminal 105 residues of BPV-1. PlasmidpTATΔcys-161 encoded tat residues 1-21 and 38-62, followed by thecarboxy-terminal 161 residues of BPV-1. We constructed plasmidspTATΔcys-105 and pTATΔcys-161 from intermediate plasmids pFTE103 andpFTE403, respectively.

We produced pFTE103 and pFTE403 (as well as pFTE501) by ligatingdifferent inserts into BamHI-cleaved (complete digestion) vector pTAT72.

To obtain the insertion fragment for pFTE103, we isolated a 929 basepair PleI-BamHI fragment from pXB314 and ligated it to a double-strandedlinker consisting of synthetic oligonucleotide FTE.3 (SEQ ID NO:23) andsynthetic oligonucleotide FTE.4 (SEQ ID NO:24). The linker encoded tatresidues 61-67 and had a BamHI overhang at the 5' end and a PleIoverhang at the 3' end. We ligated the linker-bearing fragment frompXB3314 into BamHI-cleaved pTAT72, to obtain pFTE103. To obtain theinsertion fragment for pFTE403, we digested pXB314 with NcoI and SpeI,generated blunt ends with Klenow treatment and ligated a BglII linkerconsisting of GAAGATCTTC (New England Biolabs, Beverly, Mass., Cat. No.1090) (SEQ ID NO:35) duplexed with itself. We purified the resulting822-base pair fragment by electrophoresis and then ligated it intoBamHI-digested pTAT72 vector, to obtain pFTE403.

To delete tat residues 22-37, thereby obtaining plasmid pTATΔcys-105from pFTE103 and pTATΔcys-161 from pFTE403, we employed the same method(described above) used to obtain plasmid pTATΔcys-249 from pFTE501.

TATΔcys-HPV E2 Genetic Fusions

We constructed plasmids pTATΔcys-HE2.85 and pTATΔcys-HE2.121 to encode afusion protein consisting of the tatΔcys transport moiety (tat residues1-21, 38-72) followed by the carboxy-terminal 85 or 121 residues ofHPV-16, respectively.

Our starting plasmids in the construction of pTATΔcys-HE2.85 andpTATΔcys-HE2.121 were, respectively, pET8c-85 and pET8c-123 (bothdescribed above). We digested pET8c-85 and pET8c-123 with BglII andNcoI, and isolated the large fragment in each case (4769 base pairs frompET8c-85 or 4880 base pairs from pET8c-123) for use as a vector. In bothvectors, the E2 coding regions begin at the NcoI site. Into bothvectors, we inserted the 220 bp BglII-AatII fragment from plasmidpTATΔcys, and a synthetic fragment. The 5' end of the BglII-AatIIfragment is upstream of the T7 promoter and encodes the first 40residues of tatΔcys (i.e., residues 1-21, 38-56). The synthetic fragmentconsisting of annealed oligonucleotides 374.67 (SEQ ID NO:25) and 374.68(SEQ ID NO:26), encoded tat residues 57-72, with an AatII overhang atthe 5' end and an NcoI overhang at the 3' end.

JB Series of Genetic Fusions

Plasmid pJB106 encodes a fusion protein (FIG. 23) (SEQ ID NO:38) inwhich an amino-terminal methionine residue is followed by tat residues47-58 and then HPV-16 E2 residues 245-365. To obtain pJB106, we carriedout a three-way ligation, schematically depicted in FIG. 22. Wegenerated a 4602 base pair vector fragment by digesting plasmid pET8cwith NcoI and BamHI. One insert was a 359 base pair MspI-BamHI fragmentfrom pET8c-123, encoding HPV-16 E2 residues 248-365. The other insertwas a synthetic fragment consisting of the annealed oligonucleotidepair, 374.185 (SEQ ID NO:27) and 374.186 (SEQ ID NO:28). The syntheticfragment encoded the amino-terminal methionine and tat residues 47-58,plus HPV16 residues 245-247 (i.e., ProAspThr). The synthetic fragmenthad an NcoI overhang at the 5' end and an MspI overhang at the 3' end.

We obtained plasmids pJB117 (SEQ ID NO:59), pJB118 (SEQ ID NO:60),pJB119 (SEQ ID NO:61), pJB120 (SEQ ID NO:62) and pJB122 (SEQ ID NO:63)by PCR deletion cloning in a manner similar to that used for pTATΔcys(described above and in FIG. 19). We constructed plasmids pJB117 andpJB118 by deleting segments of pTATΔcys-HE2.121. We constructed plasmidspJB119 and pJB120 by deleting segments of pTATΔcys-161. In all fourclonings, we used PCR primer 374.122 (SEQ ID NO:29) to cover the HindIIIsite downstream of the tat-E2 coding region. In each case, the otherprimer spanned the NdeI site at the start of the tatΔcys codingsequence, and deleted codons for residues at the beginning of tatΔcys(i.e., residues 2-21 and 38-46 for pJB117 and pJB119; and residues 2-21for pJB118 and pJB120). For deletion of residues 2-21, we used primer379.11 (SEQ ID NO:30). For deletion of residues 2-21 and 38-46, we usedprimer 379.12 (SEQ ID NO:31). Following the PCR reaction, we digestedthe PCR products with NdeI and HindIII. We then cloned the resultingrestriction fragments into vector pTATΔcys-HE2.121, which had beenpreviously digested with NdeI plus HindIII to yield a 4057 base pairreceptor fragment. Thus, we constructed expression plasmids encodingfusion proteins consisting of amino acid residues as follows:

JB117=Tat47-72-HPV16 E2 245-365;

JB118=Tat38-72-HPV16 E2 245-365;

JB119=Tat47-62-BPV1 E2 250-410; and

JB120=Tat38-62-BPV1 E2 250-410.

We constructed pJB122, encoding tat residues 38-58 followed by HPV16 E2residues 245-365 (i.e., the E2 carboxy-terminal 121 amino acids), bydeleting from pJB118 codons for tat residues 59-72. We carried out thisdeletion by PCR, using primer 374.13 (SEQ ID NO:32), which covers theAatII site within the tat coding region, and primer 374.14 (SEQ IDNO:33), which covers the AatII site slightly downstream of the uniqueHindIII site downstream of the Tat-E2 gene. We digested the PCR productwith AatII and isolated the resulting restriction fragment. In the finalpJB122 construction step, we inserted the isolated AatII fragment intoAatII-digested vector pJB118.

It should be noted that in all five of our pJB constructs describedabove, the tat coding sequence was preceded by a methionine codon forinitiation of translation.

Purification of Tat-E2 Fusion Proteins

In all cases, we used E.coli to express our tat-E2 genetic fusions. Ourgeneral procedure for tat-E2 protein purification included the followinginitial steps: pelleting the cells; resuspending them in 8-10 volumes oflysis buffer (25 mM Tris (pH 7.5), 25 mM NaCl, 1 mM DTT, 0.5 mM EDTA)containing protease inhibitors--generally, 1 mM PMSF, 4 μg/ml E64, 50μml aprotinin, 50 μg/ml pepstatin A, and 3 mM benzamidine); lysing thecells in a French press (2 passes at 12,000 psi); and centrifuging thelysates at 10,000-12,000×g for 1 hour (except FTE proteins), at 4° C.Additional steps employed in purifying particular tat-E2 fusion proteinsare described below.

E2.103 and E2.249--Following centrifugation of the lysate, we loaded thesupernatant onto a Fast S Sepharose column and eluted the E2.103 orE2.249 protein with 1M NaCl. We then further purified the E2.103 orE2.249 by chromatography on a P60 gel filtration column equilibratedwith 100 mM HEPES (pH 7.5), 0.1 mM EDTA and 1 mM DTT.

FTE103--Following centrifugation of the lysate at 10,000×g for 10 min.at 4° C., we recovered the FTE103 protein (which precipitated) byresuspending the pellet in 6M urea and adding solid guanidine-HCl to afinal concentration of 7M. After centrifuging the suspension, wepurified the FTE103 protein from the supernatant by chromatography on anA.5M gel filtration column in 6M guanidine, 50 mM sodium phosphate (pH5.4), 10 mM DTT. We collected the FTE103-containing fractions from thegel filtration column according to the appearance of a band having anapparent molecular weight of 19 kDa on Coomassie-stained SDSpolyacrylamide electrophoresis gels.

FTE403--Our purification procedure for FTE403 was essentially the sameas that for FTE103, except that FTE403 migrated on the gel filtrationcolumn with an apparent molecular weight of 25 kDa.

FTE501--Following centrifugation of the lysate at 10,000×g, for 30minutes, we resuspended the pellet in 6M urea, added solid guanidine-HClto a final concentration of 6M, and DTT to a concentration of 10 mM.After 30 minutes at 37° C., we clarified the solution by centrifugationat 10,000×g for 30 minutes. We then loaded the sample onto an A.5agarose gel filtration column in 6M guanidine-HCl, 50 mM sodiumphosphate (pH 5.4), 10 mM DTT and collected the FTE501-containingfractions from the gel filtration column, according to the appearance ofa band having an apparent molecular weight of 40 kDa onCoomassie-stained SDS polyacrylamide electrophoresis gels. We loaded thegel filtration-purified FTE501 onto a C₁₈ reverse phase HPLC column andeluted with a gradient of 0-75% acetonitrile in 0.1% trifluoroaceticacid. We collected the FTE501 protein in a single peak with an apparentmolecular weight of 40 kDa.

TatΔcys-105--Following centrifugation of the lysate, we loaded thesupernatant onto a Q-Sepharose column equilibrated with 25 mM Tris (pH7.5), 0.5 mM EDTA. We loaded the Q-Sepharose column flow-through onto anS-Sepharose column equilibrated with 25 mM MES (pH 6.0), after adjustingthe Q-Sepharose column flow-through to about pH 6.0 by adding MES (pH6.0) to a final concentration of 30 mM. We recovered the tatΔcys-105protein from the S-Sepharose column by application of sequential NaClconcentration steps in 25 mM MES (pH 6.0). TatΔcys-105 eluted in the pH6.0 buffer at 800-1000 mM NaCl.

TatΔcys-161--Following centrifugation of the lysate, we loaded thesupernatant onto an S-Sepharose column equilibrated with 25 mM Tris (pH7.5), 0.5 mM EDTA. We recovered the tatΔcys-161 from the S-Sepharosecolumn by application of a NaCl step gradient in 25 mM Tris (pH 7.5).TatΔcys-161 eluted in the pH 7.5 buffer at 500-700 mM NaCl.

TatΔcys-249--Following centrifugation of the lysate, we loaded thesupernatant onto a Q-Sepharose column equilibrated with 25 mM Tris (pH7.5), 0.5 mM EDTA. We recovered the tatΔcys-249 from the S-Sepharosecolumn by application of a NaCl step gradient in 25 mM Tris (pH 7.5).TatΔcys-249 eluted in the 600-800 mM portion of the NaCl step gradient.

TatΔcys-HE2.85 and TatΔcys-HE2.121--Following centrifugation of thelysate, we loaded the supernatant onto a Q-Sepharose column. We loadedthe flow-through onto an S-Sepharose column. We recovered thetatΔcys-HE2.85 or tatΔcys-HE2.121 from the S-Sepharose column byapplication of a NaCl step gradient. Both proteins eluted with 1M NaCl.

HPV E2 and HPV E2CCSS--See Example 9 (above).

JB106--Following centrifugation of the lysate, and collection of thesupernatant, we added NaCl to 300 mM. We loaded the supernatant withadded NaCl onto an S-Sepharose column equilibrated with 25 mM HEPES (pH7.5). We treated the column with sequential salt concentration steps in25 mM HEPES (pH 7.5), 1.5 mM EDTA, 1 mM DTT. We eluted the JB106 proteinfrom the S-Sepharose column with 1M NaCl.

JB117--Following centrifugation of the lysate, and collection of thesupernatant, we added NaCl to 300 mM. Due to precipitation of JB117 at300 mM NaCl, we diluted the JB117 supernatant to 100 mM NaCl andbatch-loaded the protein onto the S-Sepharose column. We eluted JB117from the S-Sepharose column with 1M NaCl in 25 mM Tris (pH 7.5), 0.3 mMDTT.

JB118--Following centrifugation of the lysate, and collection of thesupernatant, we added NaCl to 300 mM. We loaded the supernatant withadded NaCl onto an S-Sepharose column equilibrated with 25 mM Tris (pH7.5). We eluted the JB118 protein from the S-Sepharose column with 1MNaCl in 25 mM Tris (pH 7.5), 0.3 mM DTT.

JB119, JB120, JB121 and JB122--Following centrifugation of the lysate,and collection of the supernatant, we added NaCl to 150 mM for JB119 andJB121, and 200 mM for JB120 and JB122. We loaded the supernatant withadded NaCl onto an S-Sepharose column equilibrated with 25 mM Tris (pH7.5). We eluted proteins JB119, JB120, JB121 and JB122 from theS-Sepharose column with 1M NaCl in 25 mM Tris (pH 7.5), 0.3 mM DTT.

EXAMPLE 14 E2 Repression Assays--Additional Conjugates

We tested our tat-E2 fusion proteins for inhibition of transcriptionalactivation by the full-length papillomavirus E2 protein ("repression").We measured E2 repression with a transient co-transfection assay in COS7cells. The COS7 cells used in this assay were maintained in culture foronly short periods of time. We thawed the COS7 cells at passage 13 andused them only through passage 25. Long periods of propagation led tolow levels of E2 transcriptional activation and decreased repression andreproducibility. Our repression assay and method of computing repressionactivity are described in Example 9 (above). For the conjugatesTxE2.103, TxE22.249, FTE103, FTE202, FTE403 and FTE501, we substitutedthe BPV-1 E2 transactivator, in equal amount, for the HPV-16 E2transactivator. Accordingly, instead of transfecting with the HPV-16 E2expression plasmid pAHE2, we transfected with the BPV-1 E2 expressionplasmid pXB323, which is fully described in U.S. Pat. No. 5,219,990.

The genetic fusion protein JB106 has consistently been our most potenttat-E2 repressor conjugate. Data from a repression assay comparing JB106and TxHE2CCSS are shown in Table III. FIG. 24 graphically depicts theresults presented in Table III.

In addition to JB106, several other tat-E2 repressor conjugates haveyielded significant repression. As shown in Table IV, TxHE2, TxHE2CCSS,JB117, JB118, JB119, JB120 and JB122 displayed repression levels in the++ range.

                  TABLE III                                                       ______________________________________                                        Protein             average of                                                                              average                                                                              %                                        added (μg/ml)                                                                        cpm-bkgd* duplicates                                                                              cpm-bkgd                                                                             repression                               ______________________________________                                        0              3,872                                                          0              3,694      3783  --     --                                     0             17,896                                                          0             18,891    18,393  14,610 --                                     1   JB106     16,384                                                          1   JB106     17,249    16,816  13,033 10.8                                   3   JB106     11,456                                                          3   JB106     10,550    11,003   7,220 50.6                                   10  JB106      6,170                                                          10  JB106      7,006     6,588   2,805 81.0                                   30  JB106      4,733                                                          30  JB106      4,504     4,618    835  94.3                                   1   TxHE2CCSS 17,478                                                          1   TxHE2CCSS 18,041    17,762  13,979 4.3                                    3   TxHE2CCSS 14,687                                                          3   TxHE2CCSS 15,643    15,165  11,382 22.1                                   10  TxHE2CCSS 12,914                                                          10  TxHE2CCSS 12,669    12,791   9,008 38.3                                   30  TxHE2CCSS  7,956                                                          30  TxHE2CCSS  8,558     8,257   4,474 69.4                                   1   HE2.123   18,290                                                          1   HE2.123   18,744    18,517  14,734 0                                      3   HE2.123   17,666                                                          3   HE2.123   18,976    18,321  14,538 1.3                                    10  HE2.123   18,413                                                          10  HE2.123   17,862    18,137  14,354 2.6                                    30  HE2.123   18,255                                                          30  HE2.123   18,680    18,467  14,684 0.3                                    ______________________________________                                         *Bkgd = 158 cpm.                                                         

Table IV summarizes our tat-E2 repressor assay results. Although wetested all of our tat-E2 repressor conjugates in similar assays, theconjugates were not all simultaneously tested in the same assay.Accordingly, we have expressed the level of repression activity,semi-quantitatively, as +++, ++, +, ±, or -, with +++ being strongrepression, and - being no detectable repression. FIG. 24 illustratesthe repression activity rating system used in Table IV. JB106exemplifies the +++ activity level. TxHE2CCSS exemplifies the ++activity level. The negative control, HE2.123, exemplifies the -activity level. The + activity level is intermediate between theactivity observed with TxHE2CCSS and HE2.123. The two conjugates whoseactivity is shown as ± had weak (but detectable) activity in some assaysand no detectable activity in other assays.

                  TABLE IV                                                        ______________________________________                                                                          Repression                                  Protein   Tat residues                                                                              E2 residues Level                                       ______________________________________                                        TxE2.103  37-72       BPV-1 308-410                                                                             +                                           TxE2.249  37-72       BPV-1 162-410                                                                             -                                           TxHE2     37-72       HPV-16 245-365                                                                            ++                                          TxHE2CCSS 37-72       HPV-16 245-365                                                                            ++                                          FTE103     1-67       BPV-1 306-410                                                                             -                                           FTE208     1-62       BPV-1 311-410                                                                             -                                           FTE403     1-62       BPV-1 250-410                                                                             -                                           FTE501     1-62       BPV-1 162-410                                                                             -                                           TatΔcys-                                                                            1-21,     BPV-1 306-410                                                                             -                                           105       38-67                                                               TatΔcys-                                                                            1-21,     BPV-1 250-410                                                                             +/-                                         161       38-62                                                               TatΔcys-                                                                            1-21,     BPV-1 162-410                                                                             +/-                                         249       38-62                                                               TatΔcys-                                                                            1-21,     HPV-16 281-365                                                                            +                                           HE2.85    38-72                                                               TatΔcys-                                                                            1-21,     HPV-16 245-365                                                                            +                                           HE2.121   38-72                                                               JB106     47-58       HPV-16 245-365                                                                            +++                                         JB117     47-72       HPV-16 245-365                                                                            ++                                          JB118     38-72       HPV-16 245-365                                                                            ++                                          JB119     47-62       BPV-1 250-410                                                                             ++                                          JB120     38-62       BPV-1 250-410                                                                             ++                                          JB122     38-58       HPV-16 245-365                                                                            ++                                          ______________________________________                                    

FTE103, FTE403, FTE208 and FTE501, the four conjugates having the tatamino-terminal region (i.e., residues 1-21) and the cysteine-rich region(i.e., residues 22-37) were completely defective for repression. Sincewe have shown, by indirect immunofluorescence, that FTE501 enters cells,we consider it likely that the E2 repressor activity has been lost inthe FTE series as a result of the linkage to the tat transportpolypeptide. Our data show that the absence of the cysteine-rich regionof the tat moiety generally increased E2 repressor activity. Inaddition, the absence of the cysteine-rich region in tat-E2 conjugatesappeared to increase protein production levels in E.coli, and increaseprotein solubility, without loss of transport into target cells.Deletion of the amino-terminal region of tat also increased E2 repressoractivity. Fusion protein JB106, with only tat residues 47-58, was themost potent of our tat-E2 repressor conjugates. However, absence of thetat cysteine-rich region does not always result in preservation of E2repressor activity in the conjugate. For example, the chemical conjugateTxE2.249 was insoluble and toxic to cells. Thus, linkage of even acysteine-free portion of tat may lead to a non-functional E2 repressorconjugate.

EXAMPLE 15 Cleavable E2 Conjugates

Chemical conjugation of tat moieties to E2 protein resulted in at leasta 20-fold reduction in binding of the E2 protein to E2 binding sites onDNA (data not shown). Therefore, we conducted experiments to evaluatecleavable cross-linking between the tat transport moiety and the E2repressor moiety. We tested various cleavable cross-linking methods.

In one series of experiments, we activated the cysteine sulfhydrylgroups of HPV E2-CCSS protein with aldrithiol in 100 mM HEPES (pH 7.5),500 mM NaCl. We isolated the activated E2 repressor by gel filtrationchromatography and treated it with tat37-72. We achieved lowcross-linking efficiency because of rapid E2-CCSS dimer formation upontreatment with aldrithiol. To avoid this problem, we put the E2-CCSSinto 8M urea, at room temperature, and treated it with aldrithiol at 23°C. for 60 minutes under denaturing conditions. We then refolded theE2CCSS-aldrithiol adduct, isolated it by gel filtration chromatography,and then allowed it to react with tat37-72. This procedure resulted inexcellent cross-linking. We also cross-Linked E2CSSS and E2CCSC totat37-72, using a modification of the urea method, wherein we usedS-Sepharose chromatography instead of gel filtration to isolate theE2-aldrithiol adducts. This modification increased recovery of theadducts and resulted in cross-linkage of approximately 90% of the E2starting material used in the reaction.

The cleavable tat-E2 conjugates exhibited activity in the repressionassay. However, the repression activity of the cleavable conjugates wasslightly lower than that of similar conjugates cross-linkedirreversibly. The slightly lower activity of the cleavable conjugatesmay be a reflection of protein half-life in the cells. Tat is relativelystable in cells. E2 proteins generally have short half-lives in cells.Thus, irreversible cross-linkage between a tat moiety and an E2 moietymay stabilize the E2 moiety.

EXAMPLE 16 Herpes Simplex Virus Repressor Conjugate

Herpes simplex virus ("HSV") encodes a transcriptional activator, VP16,which induces expression of the immediate early HSV genes. Friedman etal. have produced an HSV VP16 repressor by deleting the carboxy-terminaltransactivation domain of VP16 ("Expression of a Truncated ViralTrans-Activator Selectively Impedes Lytic Infection by Its CognateVirus", Nature, 335, pp. 452-54 (1988)). We have produced an HSV-2 VP16repressor in a similar manner.

To test cellular uptake and VP16 repressor activity of transportpolypeptide-VP16 repressor conjugates, we simultaneously transfected aVP16-dependent reporter plasmid and a VP16 repressor plasmid into COS7cells. Then we exposed the transfected cells to a transportpolypeptide-VP16 repressor conjugate or to an appropriate control. Therepression assay, described below, was analogous to the E2 repressionassay described above, in Example 9.

VP16 Repression Assay Plasmids

Our reporter construct for the VP16 repression assay was plasmidp175kCAT, obtained from G. Hayward (see, P. O'Hare and G. S. Hayward,"Expression of Recombinant Genes Containing Herpes Simplex VirusDelayed-Early and Immediate-Early Regulatory Regions and TransActivation by Herpes Virus Infection", J. Virol., 52, pp. 522-31(1984)). Plasmid p175kCAT contains the HSV-1 IE175 promoter driving aCAT reporter gene.

Our HSV-2 transactivator construct for the VP16 repression assay wasplasmid pXB324, which contained the wild-type HSV-2 VP16 gene under thecontrol of the chicken β-actin promoter. We constructed pXB324 byinserting into pXB100 (P. Han et al., "Transactivation of HeterologousPromoters by HIV-1 Tat", Nuc. Acids Res., 19, pp. 7225-29 (1991)),between the XhoI site and BamHI site, a 280 base pair fragmentcontaining the chicken β-actin promoter and a 2318 base pair BamHI-EcoRIfragment from plasmid pCA5 (O'Hare and Hayward, supra) encoding theentire wild type HSV-2 VP16 protein.

Tat-VP16 Repressor Fusion Protein

We produced in bacteria fusion protein tat-VP16R.GF (SEQ ID NO:58),consisting of amino acids 47-58 of HIV tat protein followed by aminoacids 43-412 of HSV VP16 protein. For bacterial production of a tat-VP16repressor fusion protein, we constructed plasmid pET/tat-VP16R.GF, in athree-piece ligation. The first fragment was the vector pET-3d(described above under the alternate designation "pET-8c") digested withNcoI and BglII (approximately 4600 base pairs). The second fragmentconsisted of synthetic oligonucleotides 374.219 (SEQ ID NO:39) and374.220 (SEQ ID NO:40), annealed to form a double-stranded DNA molecule.The 5' end of the synthetic fragment had an NcoI overhang containing anATG translation start codon. Following the start codon were codons fortat residues 47-58. Immediately following the tat codons, in frame, werecodons for VP16 residues 43-47. The 3' terminus of the syntheticfragment was a blunt end for ligation to the third fragment, an 1134base pair PvuII-BglII fragment from pXB324R4, containing codons 48-412of HSV-2 VP16. We derived pXB324R4 from pXB324 (described above).Plasmid pXB324R2 was an intermediate in the construction of pXB324R4.

We constructed pXB324R2 by inserting into pXB100 a 1342 base pairBamHI-AatII fragment, from pXB324, encoding the N-terminal 419 aminoacids of HSV-2 VP16. To provide an in-frame stop codon, we used a 73base pair AatII-EcoRI fragment from pSV2-CAT (C. M. Gorman et al.,Molecular & Cellular Biology, 2, pp. 1044-51 (1982)). Thus, pXB324R2encoded the first 419 amino acids of HSV-2 VP16 and an additional sevennon-VP16 amino acids preceding the stop codon. To construct pXB324R4, wecarried out a 3-piece ligation involving a 5145 base pair MluI-EcoRIfragment from pXB324R2, and two insert fragments. One insert was a 115base pair MluI-NspI fragment from pXB324R2, encoding the first 198residues of VP16. The second insert fragment was a double-strandedsynthetic DNA molecule consisting of the synthetic oligonucleotides374.32 (SEQ ID NO:41) and 374.33 (SEQ ID NO:42). When annealed, theseoligonucleotides formed a 5' NspI sticky end and a 3' EcoRI sticky end.This synthetic fragment encoded VP16 residues 399-412, followed by atermination codon. Thus, plasmid pXB324R4 differed from pXB324R2 bylacking codons for VP16 amino acids 413-419 and the seven extraneousamino acids preceding the stop codon.

Purification of tat-VP16R.GF Fusion Protein

We expressed our genetic construct for tat-VP16R.GF in E.coli. Weharvested the transformed E.coli by centrifugation; resuspended thecells in 8-10 volumes of lysis buffer (25 mM Tris (pH 7.5), 25 mM NaCl,1 mM DTT, 0.5 mM EDTA, 1 mM PMSF, 4 μg/ml E64, 50 μg/ml aprotinin, 50μml pepstatin A, and 3 mM benzamidine); lysed the cells in a Frenchpress (2 passes at 12,000 psi); and centrifuged the lysate at 10,000 to12,000×g for 1 hour, at 4° C. Following centrifugation of the lysate, weloaded the supernatant onto a Fast Q-Sepharose column equilibrated with25 mM Tris (pH 7.5), 0.5 mM EDTA. We loaded the Q-Sepharose flow-throughonto a Fast S-Sepharose column equilibrated in 25 mM MES (pH 6.0), 0.1mM EDTA, 2 mM DTT. We recovered the tat-VP16 fusion protein from theS-Sepharose column with sequential NaCl concentration steps in 25 mM MES(pH 6.0), 0.1 mM EDTA, 2 mM DTT. The tat-VP16 fusion protein eluted inthe 600-1000 mM NaCl fractions.

VP16 Repression Assay

We seeded HeLa cells in 24-well culture plates at 10⁵ cells/well. Thefollowing day, we transfected the cells, using the DEAE-dextran method,as described by B. R. Cullen, "Use of Eukaryotic Expression Technologyin the Functional Analysis of Cloned Genes", Meth. Enzymol., vol. 152,p. 684 (1987). We precipitated the DNA for the transfections andredissolved it, at a concentration of approximately 100 μml, in 100 mMNaCl, 10 mM Tris (pH 7.5). For each transfection, the DNA-DEAE mixconsisted of: 200 ng p175kCAT (±1 ng pXB324) or 200 ng pSV-CAT(control), 1 mg/ml DEAE-dextran, and PBS, to a final volume of 100 μl.We exposed the cells to this mixture for 15-20 minutes, at 37° C., withoccasional rocking of the culture plates. We then added to each well, 1ml fresh DC medium (DMEM+10% serum) with 80 μM chloroquine. Afterincubating the cells at 37° C. for 2.5 hours, we aspirated the mediumfrom each well and replaced it with fresh DC containing 10% DMSO. After2.5 minutes at room temperature, we aspirated the DMSO-containing mediumand replaced it with fresh DC containing 0, 10 or 50 μg/ml purifiedtat-VP16.GF. The following day, we replaced the medium in each well withfresh medium of the same composition. Twenty-four hours later, we lysedthe HeLa cells with 0.65% NP-40 (detergent) in 10 mM Tris (pH 8.0), 1 mMEDTA, 150 mM NaCl. We measured the protein concentration in eachextract, for sample normalization in the assay.

At a tat-VP16.GF concentration of 50 μml, cellular toxicity interferedwith the assay. At a concentration of 10 μg/ml, the tat-VP16.GF fusionprotein yielded almost complete repression of VP16-dependent CATexpression, with no visible cell death and approximately 30% repressionof non-VP16-dependent CAT expression in controls. Thus, we observedspecific repression of VP16-dependent transactivation in addition to alesser amount non-specific repression.

EXAMPLE 17 Transport polypeptide--DNA Conjugates

Transcriptional activation by a DNA-binding transcription factor can beinhibited by introducing into cells DNA having the binding site for thattranscription factor. The transcription factor becomes bound by theintroduced DNA and is rendered unavailable to bind at the promoter sitewhere it normally functions. This strategy has been employed to inhibittranscriptional activation by NF-κB (Bielinska et al., "Regulation ofGene Expression with Double-Stranded Phosphorothioate Oligonucleotides",Science, vol. 250, pp. 997-1000 (1990)). Bielinska et al. observeddose-dependent inhibition when the double stranded DNA was put in thecell culture medium. We conjugated the transport polypeptide tat 37-72to the double stranded DNA molecule to determine whether suchconjugation would enhance the inhibition by increasing the cellularuptake of the DNA.

We purchased four custom-synthesized 39-mer phosphorothioateoligonucleotides designated NF1, NF2, NF3 and NF4, having nucleotidesequences (SEQ ID NO:43), (SEQ ID NO:44), (SEQ ID NO:45) and (SEQ IDNO:46), respectively. NF1 and NF2 form a duplex corresponding to thewild type NF-κB binding site. NF3 and NF4 form a duplex corresponding toa mutant NF-κB binding site.

We dissolved NF1 and NF3 in water, at a concentration of approximately 4mg/ml. We then put 800 μg of NF1 and NF3 separately into 400 μl of 50 mMtriethanolamine (pH 8.2), 50 mM NaCl, 10 mM Traut's reagent. We allowedthe reaction to proceed for 50 minutes at room temperature. We stoppedthe reaction by gel filtration on a P6DG column (BioRad, Richmond,Calif.) equilibrated with 50 mM HEPES (pH 6.0), 50 mM NaCl, to removeexcess Traut's reagent. We monitored 260 nm absorbance to identify theoligonucleotide-containing fractions. Our recovery of theoligonucleotides was approximately 75%. We then annealed Traut-modifiedNF1 with NF2 (0.55 mg/ml final concentration) and annealedTraut-modified NF3 with NF4 0.50 mg/ml final concentration). Finally, weallowed 0.4 mg of each Traut-modified DNA to react with 0.6 mg oftat37-72-BMH (prepared as described in Example 9, above), in 1 ml of 100mM HEPES (pH 7.5), for 60 minutes at room temperature. We monitored theextent of the cross-linking reaction by polyacrylamide gelelectrophoresis followed by ethidium bromide staining of the gel. Ingeneral, we observed that about 50% of the DNA was modified under theseconditions.

These double-stranded DNA molecules were tested, essentially accordingto the methods of Bielinska et al. (supra), with and without tatlinkage, for inhibition of NF-κB transcriptional activation. Tat linkagesignificantly enhanced the inhibition of by NF-κB.

Recombinant DNA sequences prepared by the processes described herein areexemplified by a culture deposited in the American Type CultureCollection, Rockville, Md. The Escherichia coli culture identified aspJB106 was deposited on Jul. 28, 1993 and assigned ATCC accession number69368.

While we have described a number of embodiments of this invention, it isapparent that our basic constructions can be altered to provide otherembodiments that utilize the processes and products of this invention.Therefore, it will be appreciated that the scope of this invention is tobe defined by the appended claims rather than by the specificembodiments that have been presented by way of example.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 69                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 86 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: human immunodeficiency virus                                    (B) STRAIN: type 1                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       MetGluProValAspProArgLeuGluProTrpLysHisProGlySer                              151015                                                                        GlnProLysThrAlaCysThrAsnCysTyrCysLysLysCysCysPhe                              202530                                                                        HisCysGlnValCysPheIleThrLysAlaLeuGlyIleSerTyrGly                              354045                                                                        ArgLysLysArgArgGlnArgArgArgProProGlnGlySerGlnThr                              505560                                                                        HisGlnValSerLeuSerLysGlnProThrSerGlnSerArgGlyAsp                              65707580                                                                      ProThrGlyProLysGlu                                                            85                                                                            (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 36 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       CysPheIleThrLysAlaLeuGlyIleSerTyrGlyArgLysLysArg                              151015                                                                        ArgGlnArgArgArgProProGlnGlySerGlnThrHisGlnValSer                              202530                                                                        LeuSerLysGln                                                                  35                                                                            (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       CysPheIleThrLysAlaLeuGlyIleSerTyrGlyArgLysLysArg                              151015                                                                        ArgGlnArgArgArgPro                                                            20                                                                            (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       PheIleThrLysAlaLeuGlyIleSerTyrGlyArgLysLysArgArg                              151015                                                                        GlnArgArgArgProGlyGlyCys                                                      20                                                                            (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       CysGlyGlyTyrGlyArgLysLysArgArgGlnArgArgArgPro                                 151015                                                                        (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       TyrGlyArgLysLysArgArgGlnArgArgArgProGlyGlyCys                                 151015                                                                        (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 56 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       MetGluProValAspProArgLeuGluProTrpLysHisProGlySer                              151015                                                                        GlnProLysThrAlaPheIleThrLysAlaLeuGlyIleSerTyrGly                              202530                                                                        ArgLysLysArgArgGlnArgArgArgProProGlnGlySerGlnThr                              354045                                                                        HisGlnValSerLeuSerLysGln                                                      5055                                                                          (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 39 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       GATCCCAGACCCACCAGGTTTCTCTGTCGGGCCCTTAAG39                                     (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 39 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       AATTCTTAAGGGCCCGACAGAGAAACCTGGTGGGTCTGG39                                     (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5098 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      TTGAAGACGAAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGATAATAAT60                GGTTTCTTAGACGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTT120               ATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCT180               TCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCC240               CTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAA300               AGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGG360               TAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGT420               TCTGCTATGTGGCGCGGTATTATCCCGTGTTGACGCCGGGCAAGAGCAACTCGGTCGCCG480               CATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTAC540               GGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGC600               GGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAA660               CATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACC720               AAACGACGAGCGTGACACCACGATGCCTGCAGCAATGGCAACAACGTTGCGCAAACTATT780               AACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGA840               TAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAA900               ATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAA960               GCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAA1020              TAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGT1080              TTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGT1140              GAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTG1200              AGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGT1260              AATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCA1320              AGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATAC1380              TGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTAC1440              ATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCT1500              TACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGG1560              GGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACA1620              GCGTGAGCATTGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGT1680              AAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTA1740              TCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTC1800              GTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGC1860              CTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAA1920              CCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAG1980              CGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTTCTCCTTACGCATCT2040              GTGCGGTATTTCACACCGCATATATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCA2100              TAGTTAAGCCAGTATACACTCCGCTATCGCTACGTGACTGGGTCATGGCTGCGCCCCGAC2160              ACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACA2220              GACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGA2280              AACGCGCGAGGCAGCTGCGGTAAAGCTCATCAGCGTGGTCGTGAAGCGATTCACAGATGT2340              CTGCCTGTTCATCCGCGTCCAGCTCGTTGAGTTTCTCCAGAAGCGTTAATGTCTGGCTTC2400              TGATAAAGCGGGCCATGTTAAGGGCGGTTTTTTCCTGTTTGGTCACTTGATGCCTCCGTG2460              TAAGGGGGAATTTCTGTTCATGGGGGTAATGATACCGATGAAACGAGAGAGGATGCTCAC2520              GATACGGGTTACTGATGATGAACATGCCCGGTTACTGGAACGTTGTGAGGGTAAACAACT2580              GGCGGTATGGATGCGGCGGGACCAGAGAAAAATCACTCAGGGTCAATGCCAGCGCTTCGT2640              TAATACAGATGTAGGTGTTCCACAGGGTAGCCAGCAGCATCCTGCGATGCAGATCCGGAA2700              CATAATGGTGCAGGGCGCTGACTTCCGCGTTTCCAGACTTTACGAAACACGGAAACCGAA2760              GACCATTCATGTTGTTGCTCAGGTCGCAGACGTTTTGCAGCAGCAGTCGCTTCACGTTCG2820              CTCGCGTATCGGTGATTCATTCTGCTAACCAGTAAGGCAACCCCGCCAGCCTAGCCGGGT2880              CCTCAACGACAGGAGCACGATCATGCGCACCCGTGGCCAGGACCCAACGCTGCCCGAGAT2940              GCGCCGCGTGCGGCTGCTGGAGATGGCGGACGCGATGGATATGTTCTGCCAAGGGTTGGT3000              TTGCGCATTCACAGTTCTCCGCAAGAATTGATTGGCTCCAATTCTTGGAGTGGTGAATCC3060              GTTAGCGAGGTGCCGCCGGCTTCCATTCAGGTCGAGGTGGCCCGGCTCCATGCACCGCGA3120              CGCAACGCGGGGAGGCAGACAAGGTATAGGGCGGCGCCTACAATCCATGCCAACCCGTTC3180              CATGTGCTCGCCGAGGCGGCATAAATCGCCGTGACGATCAGCGGTCCAGTGATCGAAGTT3240              AGGCTGGTAAGAGCCGCGAGCGATCCTTGAAGCTGTCCCTGATGGTCGTCATCTACCTGC3300              CTGGACAGCATGGCCTGCAACGCGGGCATCCCGATGCCGCCGGAAGCGAGAAGAATCATA3360              ATGGGGAAGGCCATCCAGCCTCGCGTCGCGAACGCCAGCAAGACGTAGCCCAGCGCGTCG3420              GCCGCCATGCCGGCGATAATGGCCTGCTTCTCGCCGAAACGTTTGGTGGCGGGACCAGTG3480              ACGAAGGCTTGAGCGAGGGCGTGCAAGATTCCGAATACCGCAAGCGACAGGCCGATCATC3540              GTCGCGCTCCAGCGAAAGCGGTCCTCGCCGAAAATGACCCAGAGCGCTGCCGGCACCTGT3600              CCTACGAGTTGCATGATAAAGAAGACAGTCATAAGTGCGGCGACGATAGTCATGCCCCGC3660              GCCCACCGGAAGGAGCTGACTGGGTTGAAGGCTCTCAAGGGCATCGGTCGACGCTCTCCC3720              TTATGCGACTCCTGCATTAGGAAGCAGCCCAGTAGTAGGTTGAGGCCGTTGAGCACCGCC3780              GCCGCAAGGAATGGTGCATGCAAGGAGATGGCGCCCAACAGTCCCCCGGCCACGGGGCCT3840              GCCACCATACCCACGCCGAAACAAGCGCTCATGAGCCCGAAGTGGCGAGCCCGATCTTCC3900              CCATCGGTGATGTCGGCGATATAGGCGCCAGCAACCGCACCTGTGGCGCCGGTGATGCCG3960              GCCACGATGCGTCCGGCGTAGAGGATCGAGATCTCGATCCCGCGAAATTAATACGACTCA4020              CTATAGGGAGACCACAACGGTTTCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAG4080              ATATACATATGGAACCGGTCGACCCGCGTCTGGAACCATGGAAACACCCCGGGTCCCAGC4140              CGAAAACCGCGTGCACCAACTGCTACTGCAAAAAATGCTGCTTCCACTGCCAGGTTTGCT4200              TCATCACCAAAGCCCTAGGTATCTCTTACGGCCGTAAAAAACGTCGTCAGCGACGTCGTC4260              CGCCGCAGGGATCCCAGACCCACCAGGTTTCTCTGTCGGGCCCGGCGGACAGCGGCGACG4320              CCCTGCTGGAGCGCAACTATCCCACTGGCGCGGAGTTCCTCGGCGACGGCGGCGACGTCA4380              GCTTCAGCACCCGCGGCACGCAGAACTGGACGGTGGAGCGGCTGCTCCAGGCGCACCGCC4440              AACTGGAGGAGCGCGGCTATGTGTTCGTCGGCTACCACGGCACCTTCCTCGAAGCGGCGC4500              AAAGCATCGTCTTCGGCGGGGTGCGCGCGCGCAGCCAGGACCTCGACGCGATCTGGCGCG4560              GTTTCTATATCGCCGGCGATCCGGCGCTGGCCTACGGCTACGCCCAGGACCAGGAACCCG4620              ACGCACGCGGCCGGATCCGCAACGGTGCCCTGCTGCGGGTCTATGTGCCGCGCTCGAGCC4680              TGCCGGGCTTCTACCGCACCAGCCTGACCCTGGCCGCGCCGGAGGCGGCGGGCGAGGTCG4740              AACGGCTGATCGGCCATCCGCTGCCGCTGCGCCTGGACGCCATCACCGGCCCCGAGGAGG4800              AAGGCGGGCGCCTGGAGACCATTCTCGGCTGGCCGCTGGCCGAGCGCACCGTGGTGATTC4860              CCTCGGCGATCCCCACCGACCCGCGCAACGTCGGCGGCGACCTCGACCCGTCCAGCATCC4920              CCGACAAGGAACAGGCGATCAGCGCCCTGCCGGACTACGCCAGCCAGCCCGGCAAACCGC4980              CGCGCGAGGACCTGAAGTAACTGCCGCGACCGGCCGGCTCCCTTCGCAGGAGCCGGCCTT5040              CTCGGGGCCTGGCCATACATCAGGTTTTCCTGATGCCAGCCCAATCGAATATGAATTC5098                (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 4910 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      TTGAAGACGAAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGATAATAAT60                GGTTTCTTAGACGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTT120               ATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCT180               TCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCC240               CTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAA300               AGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGG360               TAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGT420               TCTGCTATGTGGCGCGGTATTATCCCGTGTTGACGCCGGGCAAGAGCAACTCGGTCGCCG480               CATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTAC540               GGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGC600               GGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAA660               CATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACC720               AAACGACGAGCGTGACACCACGATGCCTGCAGCAATGGCAACAACGTTGCGCAAACTATT780               AACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGA840               TAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAA900               ATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAA960               GCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAA1020              TAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGT1080              TTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGT1140              GAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTG1200              AGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGT1260              AATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCA1320              AGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATAC1380              TGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTAC1440              ATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCT1500              TACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGG1560              GGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACA1620              GCGTGAGCATTGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGT1680              AAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTA1740              TCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTC1800              GTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGC1860              CTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAA1920              CCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAG1980              CGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTTCTCCTTACGCATCT2040              GTGCGGTATTTCACACCGCATATATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCA2100              TAGTTAAGCCAGTATACACTCCGCTATCGCTACGTGACTGGGTCATGGCTGCGCCCCGAC2160              ACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACA2220              GACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGA2280              AACGCGCGAGGCAGCTGCGGTAAAGCTCATCAGCGTGGTCGTGAAGCGATTCACAGATGT2340              CTGCCTGTTCATCCGCGTCCAGCTCGTTGAGTTTCTCCAGAAGCGTTAATGTCTGGCTTC2400              TGATAAAGCGGGCCATGTTAAGGGCGGTTTTTTCCTGTTTGGTCACTTGATGCCTCCGTG2460              TAAGGGGGAATTTCTGTTCATGGGGGTAATGATACCGATGAAACGAGAGAGGATGCTCAC2520              GATACGGGTTACTGATGATGAACATGCCCGGTTACTGGAACGTTGTGAGGGTAAACAACT2580              GGCGGTATGGATGCGGCGGGACCAGAGAAAAATCACTCAGGGTCAATGCCAGCGCTTCGT2640              TAATACAGATGTAGGTGTTCCACAGGGTAGCCAGCAGCATCCTGCGATGCAGATCCGGAA2700              CATAATGGTGCAGGGCGCTGACTTCCGCGTTTCCAGACTTTACGAAACACGGAAACCGAA2760              GACCATTCATGTTGTTGCTCAGGTCGCAGACGTTTTGCAGCAGCAGTCGCTTCACGTTCG2820              CTCGCGTATCGGTGATTCATTCTGCTAACCAGTAAGGCAACCCCGCCAGCCTAGCCGGGT2880              CCTCAACGACAGGAGCACGATCATGCGCACCCGTGGCCAGGACCCAACGCTGCCCGAGAT2940              GCGCCGCGTGCGGCTGCTGGAGATGGCGGACGCGATGGATATGTTCTGCCAAGGGTTGGT3000              TTGCGCATTCACAGTTCTCCGCAAGAATTGATTGGCTCCAATTCTTGGAGTGGTGAATCC3060              GTTAGCGAGGTGCCGCCGGCTTCCATTCAGGTCGAGGTGGCCCGGCTCCATGCACCGCGA3120              CGCAACGCGGGGAGGCAGACAAGGTATAGGGCGGCGCCTACAATCCATGCCAACCCGTTC3180              CATGTGCTCGCCGAGGCGGCATAAATCGCCGTGACGATCAGCGGTCCAGTGATCGAAGTT3240              AGGCTGGTAAGAGCCGCGAGCGATCCTTGAAGCTGTCCCTGATGGTCGTCATCTACCTGC3300              CTGGACAGCATGGCCTGCAACGCGGGCATCCCGATGCCGCCGGAAGCGAGAAGAATCATA3360              ATGGGGAAGGCCATCCAGCCTCGCGTCGCGAACGCCAGCAAGACGTAGCCCAGCGCGTCG3420              GCCGCCATGCCGGCGATAATGGCCTGCTTCTCGCCGAAACGTTTGGTGGCGGGACCAGTG3480              ACGAAGGCTTGAGCGAGGGCGTGCAAGATTCCGAATACCGCAAGCGACAGGCCGATCATC3540              GTCGCGCTCCAGCGAAAGCGGTCCTCGCCGAAAATGACCCAGAGCGCTGCCGGCACCTGT3600              CCTACGAGTTGCATGATAAAGAAGACAGTCATAAGTGCGGCGACGATAGTCATGCCCCGC3660              GCCCACCGGAAGGAGCTGACTGGGTTGAAGGCTCTCAAGGGCATCGGTCGACGCTCTCCC3720              TTATGCGACTCCTGCATTAGGAAGCAGCCCAGTAGTAGGTTGAGGCCGTTGAGCACCGCC3780              GCCGCAAGGAATGGTGCATGCAAGGAGATGGCGCCCAACAGTCCCCCGGCCACGGGGCCT3840              GCCACCATACCCACGCCGAAACAAGCGCTCATGAGCCCGAAGTGGCGAGCCCGATCTTCC3900              CCATCGGTGATGTCGGCGATATAGGCGCCAGCAACCGCACCTGTGGCGCCGGTGATGCCG3960              GCCACGATGCGTCCGGCGTAGAGGATCGAGATCTCGATCCCGCGAAATTAATACGACTCA4020              CTATAGGGAGACCACAACGGTTTCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAG4080              ATATATATGGAACCGGTCGTTTCTCTGTCGGGCCCGGCGGACAGCGGCGACGCCCTGCTG4140              GAGCGCAACTATCCCACTGGCGCGGAGTTCCTCGGCGACGGCGGCGACGTCAGCTTCAGC4200              ACCCGCGGCACGCAGAACTGGACGGTGGAGCGGCTGCTCCAGGCGCACCGCCAACTGGAG4260              GAGCGCGGCTATGTGTTCGTCGGCTACCACGGCACCTTCCTCGAAGCGGCGCAAAGCATC4320              GTCTTCGGCGGGGTGCGCGCGCGCAGCCAGGACCTCGACGCGATCTGGCGCGGTTTCTAT4380              ATCGCCGGCGATCCGGCGCTGGCCTACGGCTACGCCCAGGACCAGGAACCCGACGCACGC4440              GGCCGGATCCGCAACGGTGCCCTGCTGCGGGTCTATGTGCCGCGCTCGAGCCTGCCGGGC4500              TTCTACCGCACCAGCCTGACCCTGGCCGCGCCGGAGGCGGCGGGCGAGGTCGAACGGCTG4560              ATCGGCCATCCGCTGCCGCTGCGCCTGGACGCCATCACCGGCCCCGAGGAGGAAGGCGGG4620              CGCCTGGAGACCATTCTCGGCTGGCCGCTGGCCGAGCGCACCGTGGTGATTCCCTCGGCG4680              ATCCCCACCGACCCGCGCAACGTCGGCGGCGACCTCGACCCGTCCAGCATCCCCGACAAG4740              GAACAGGCGATCAGCGCCCTGCCGGACTACGCCAGCCAGCCCGGCAAACCGCCGCGCGAG4800              GACCTGAAGTAACTGCCGCGACCGGCCGGCTCCCTTCGCAGGAGCCGGCCTTCTCGGGGC4860              CTGGCCATACATCAGGTTTTCCTGATGCCAGCCCAATCGAATATGAATTC4910                        (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 29 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      TATGGAACCGGTCGTTTCTCTGTCGGGCC29                                               (2) INFORMATION FOR SEQ ID NO:13:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      CGACAGAGAAACGACCGGTTCCA23                                                     (2) INFORMATION FOR SEQ ID NO:14:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 4977 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                      TTCTTGAAGACGAAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGATAAT60                AATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTG120               TTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAAT180               GCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTAT240               TCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGT300               AAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAG360               CGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAA420               AGTTCTGCTATGTGGCGCGGTATTATCCCGTGTTGACGCCGGGCAAGAGCAACTCGGTCG480               CCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCT540               TACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACAC600               TGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCA660               CAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCAT720               ACCAAACGACGAGCGTGACACCACGATGCCTGCAGCAATGGCAACAACGTTGCGCAAACT780               ATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGC840               GGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGA900               TAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGG960               TAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACG1020              AAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCA1080              AGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTA1140              GGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCA1200              CTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCG1260              CGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGA1320              TCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAA1380              TACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCC1440              TACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTG1500              TCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAAC1560              GGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCT1620              ACAGCGTGAGCATTGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCC1680              GGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTG1740              GTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATG1800              CTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCT1860              GGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGA1920              TAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCG1980              CAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTTCTCCTTACGCA2040              TCTGTGCGGTATTTCACACCGCATATATGGTGCACTCTCAGTACAATCTGCTCTGATGCC2100              GCATAGTTAAGCCAGTATACACTCCGCTATCGCTACGTGACTGGGTCATGGCTGCGCCCC2160              GACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTT2220              ACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCAC2280              CGAAACGCGCGAGGCAGCTGCGGTAAAGCTCATCAGCGTGGTCGTGAAGCGATTCACAGA2340              TGTCTGCCTGTTCATCCGCGTCCAGCTCGTTGAGTTTCTCCAGAAGCGTTAATGTCTGGC2400              TTCTGATAAAGCGGGCCATGTTAAGGGCGGTTTTTTCCTGTTTGGTCACTTGATGCCTCC2460              GTGTAAGGGGGAATTTCTGTTCATGGGGGTAATGATACCGATGAAACGAGAGAGGATGCT2520              CACGATACGGGTTACTGATGATGAACATGCCCGGTTACTGGAACGTTGTGAGGGTAAACA2580              ACTGGCGGTATGGATGCGGCGGGACCAGAGAAAAATCACTCAGGGTCAATGCCAGCGCTT2640              CGTTAATACAGATGTAGGTGTTCCACAGGGTAGCCAGCAGCATCCTGCGATGCAGATCCG2700              GAACATAATGGTGCAGGGCGCTGACTTCCGCGTTTCCAGACTTTACGAAACACGGAAACC2760              GAAGACCATTCATGTTGTTGCTCAGGTCGCAGACGTTTTGCAGCAGCAGTCGCTTCACGT2820              TCGCTCGCGTATCGGTGATTCATTCTGCTAACCAGTAAGGCAACCCCGCCAGCCTAGCCG2880              GGTCCTCAACGACAGGAGCACGATCATGCGCACCCGTGGCCAGGACCCAACGCTGCCCGA2940              GATGCGCCGCGTGCGGCTGCTGGAGATGGCGGACGCGATGGATATGTTCTGCCAAGGGTT3000              GGTTTGCGCATTCACAGTTCTCCGCAAGAATTGATTGGCTCCAATTCTTGGAGTGGTGAA3060              TCCGTTAGCGAGGTGCCGCCGGCTTCCATTCAGGTCGAGGTGGCCCGGCTCCATGCACCG3120              CGACGCAACGCGGGGAGGCAGACAAGGTATAGGGCGGCGCCTACAATCCATGCCAACCCG3180              TTCCATGTGCTCGCCGAGGCGGCATAAATCGCCGTGACGATCAGCGGTCCAGTGATCGAA3240              GTTAGGCTGGTAAGAGCCGCGAGCGATCCTTGAAGCTGTCCCTGATGGTCGTCATCTACC3300              TGCCTGGACAGCATGGCCTGCAACGCGGGCATCCCGATGCCGCCGGAAGCGAGAAGAATC3360              ATAATGGGGAAGGCCATCCAGCCTCGCGTCGCGAACGCCAGCAAGACGTAGCCCAGCGCG3420              TCGGCCGCCATGCCGGCGATAATGGCCTGCTTCTCGCCGAAACGTTTGGTGGCGGGACCA3480              GTGACGAAGGCTTGAGCGAGGGCGTGCAAGATTCCGAATACCGCAAGCGACAGGCCGATC3540              ATCGTCGCGCTCCAGCGAAAGCGGTCCTCGCCGAAAATGACCCAGAGCGCTGCCGGCACC3600              TGTCCTACGAGTTGCATGATAAAGAAGACAGTCATAAGTGCGGCGACGATAGTCATGCCC3660              CGCGCCCACCGGAAGGAGCTGACTGGGTTGAAGGCTCTCAAGGGCATCGGTCGACGCTCT3720              CCCTTATGCGACTCCTGCATTAGGAAGCAGCCCAGTAGTAGGTTGAGGCCGTTGAGCACC3780              GCCGCCGCAAGGAATGGTGCATGCAAGGAGATGGCGCCCAACAGTCCCCCGGCCACGGGG3840              CCTGCCACCATACCCACGCCGAAACAAGCGCTCATGAGCCCGAAGTGGCGAGCCCGATCT3900              TCCCCATCGGTGATGTCGGCGATATAGGCGCCAGCAACCGCACCTGTGGCGCCGGTGATG3960              CCGGCCACGATGCGTCCGGCGTAGAGGATCGAGATCTCGATCCCGCGAAATTAATACGAC4020              TCACTATAGGGAGACCACAACGGTTTCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAG4080              GAGATATACCATGGTACCAGACACCGGAAACCCCTGCCACACCACTAAGTTGTTGCACAG4140              AGACTCAGTGGACAGTGCTCCAATCCTCACTGCATTTAACAGCTCACACAAAGGACGGAT4200              TAACTGTAATAGTAACACTACACCCATAGTACATTTAAAAGGTGATGCTAATACTTTAAA4260              ATGTTTAAGATATAGATTTAAAAAGCATTGTACATTGTATACTGCAGTGTCGTCTACATG4320              GCATTGGACAGGACATAATGTAAAACATAAAAGTGCAATTGTTACACTTACATATGATAG4380              TGAATGGCAACGTGACCAATTTTTGTCTCAAGTTAAAATACCAAAAACTATTACAGTGTC4440              TACTGGATTTATGTCTATATGAGGATCCGGCTGCTAACAAAGCCCGAAAGGAAGCTGAGT4500              TGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCT4560              TGAGGGGTTTTTTGCTGAAAGGAGGAACTATATCCGGATATCCACAGGACGGGTGTGGTC4620              GCCATGATCGCGTAGTCGATAGTGGCTCCAAGTAGCGAAGCGAGCAGGACTGGGCGGCGG4680              CCAAAGCGGTCGGACAGTGCTCCGAGAACGGGTGCGCATAGAAATTGCATCAACGCATAT4740              AGCGCTAGCAGCACGCCATAGTGACTGGCGATGCTGTCGGAATGGACGATATCCCGCAAG4800              AGGCCCGGCAGTACCGGCATAACCAAGCCTATGCCTACAGCATCCAGGGTGACGGTGCCG4860              AGGATGACGATGAGCGCATTGTTAGATTTCATACACGGTGCCTGACTGCGTTAGCAATTT4920              AACTGTGATAAACTACCGCATTAAAGCTTATCGATGATAAGCTGTCAAACATGAGAA4977                 (2) INFORMATION FOR SEQ ID NO:15:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                      CTCCCATGGTACCAGACACCGGAAACC27                                                 (2) INFORMATION FOR SEQ ID NO:16:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                      GGGGGATCCTCATATAGACATAAATCC27                                                 (2) INFORMATION FOR SEQ ID NO:17:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 4977 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                      TTCTTGAAGACGAAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGATAAT60                AATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTG120               TTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAAT180               GCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTAT240               TCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGT300               AAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAG360               CGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAA420               AGTTCTGCTATGTGGCGCGGTATTATCCCGTGTTGACGCCGGGCAAGAGCAACTCGGTCG480               CCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCT540               TACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACAC600               TGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCA660               CAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCAT720               ACCAAACGACGAGCGTGACACCACGATGCCTGCAGCAATGGCAACAACGTTGCGCAAACT780               ATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGC840               GGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGA900               TAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGG960               TAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACG1020              AAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCA1080              AGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTA1140              GGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCA1200              CTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCG1260              CGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGA1320              TCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAA1380              TACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCC1440              TACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTG1500              TCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAAC1560              GGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCT1620              ACAGCGTGAGCATTGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCC1680              GGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTG1740              GTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATG1800              CTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCT1860              GGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGA1920              TAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCG1980              CAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTTCTCCTTACGCA2040              TCTGTGCGGTATTTCACACCGCATATATGGTGCACTCTCAGTACAATCTGCTCTGATGCC2100              GCATAGTTAAGCCAGTATACACTCCGCTATCGCTACGTGACTGGGTCATGGCTGCGCCCC2160              GACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTT2220              ACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCAC2280              CGAAACGCGCGAGGCAGCTGCGGTAAAGCTCATCAGCGTGGTCGTGAAGCGATTCACAGA2340              TGTCTGCCTGTTCATCCGCGTCCAGCTCGTTGAGTTTCTCCAGAAGCGTTAATGTCTGGC2400              TTCTGATAAAGCGGGCCATGTTAAGGGCGGTTTTTTCCTGTTTGGTCACTTGATGCCTCC2460              GTGTAAGGGGGAATTTCTGTTCATGGGGGTAATGATACCGATGAAACGAGAGAGGATGCT2520              CACGATACGGGTTACTGATGATGAACATGCCCGGTTACTGGAACGTTGTGAGGGTAAACA2580              ACTGGCGGTATGGATGCGGCGGGACCAGAGAAAAATCACTCAGGGTCAATGCCAGCGCTT2640              CGTTAATACAGATGTAGGTGTTCCACAGGGTAGCCAGCAGCATCCTGCGATGCAGATCCG2700              GAACATAATGGTGCAGGGCGCTGACTTCCGCGTTTCCAGACTTTACGAAACACGGAAACC2760              GAAGACCATTCATGTTGTTGCTCAGGTCGCAGACGTTTTGCAGCAGCAGTCGCTTCACGT2820              TCGCTCGCGTATCGGTGATTCATTCTGCTAACCAGTAAGGCAACCCCGCCAGCCTAGCCG2880              GGTCCTCAACGACAGGAGCACGATCATGCGCACCCGTGGCCAGGACCCAACGCTGCCCGA2940              GATGCGCCGCGTGCGGCTGCTGGAGATGGCGGACGCGATGGATATGTTCTGCCAAGGGTT3000              GGTTTGCGCATTCACAGTTCTCCGCAAGAATTGATTGGCTCCAATTCTTGGAGTGGTGAA3060              TCCGTTAGCGAGGTGCCGCCGGCTTCCATTCAGGTCGAGGTGGCCCGGCTCCATGCACCG3120              CGACGCAACGCGGGGAGGCAGACAAGGTATAGGGCGGCGCCTACAATCCATGCCAACCCG3180              TTCCATGTGCTCGCCGAGGCGGCATAAATCGCCGTGACGATCAGCGGTCCAGTGATCGAA3240              GTTAGGCTGGTAAGAGCCGCGAGCGATCCTTGAAGCTGTCCCTGATGGTCGTCATCTACC3300              TGCCTGGACAGCATGGCCTGCAACGCGGGCATCCCGATGCCGCCGGAAGCGAGAAGAATC3360              ATAATGGGGAAGGCCATCCAGCCTCGCGTCGCGAACGCCAGCAAGACGTAGCCCAGCGCG3420              TCGGCCGCCATGCCGGCGATAATGGCCTGCTTCTCGCCGAAACGTTTGGTGGCGGGACCA3480              GTGACGAAGGCTTGAGCGAGGGCGTGCAAGATTCCGAATACCGCAAGCGACAGGCCGATC3540              ATCGTCGCGCTCCAGCGAAAGCGGTCCTCGCCGAAAATGACCCAGAGCGCTGCCGGCACC3600              TGTCCTACGAGTTGCATGATAAAGAAGACAGTCATAAGTGCGGCGACGATAGTCATGCCC3660              CGCGCCCACCGGAAGGAGCTGACTGGGTTGAAGGCTCTCAAGGGCATCGGTCGACGCTCT3720              CCCTTATGCGACTCCTGCATTAGGAAGCAGCCCAGTAGTAGGTTGAGGCCGTTGAGCACC3780              GCCGCCGCAAGGAATGGTGCATGCAAGGAGATGGCGCCCAACAGTCCCCCGGCCACGGGG3840              CCTGCCACCATACCCACGCCGAAACAAGCGCTCATGAGCCCGAAGTGGCGAGCCCGATCT3900              TCCCCATCGGTGATGTCGGCGATATAGGCGCCAGCAACCGCACCTGTGGCGCCGGTGATG3960              CCGGCCACGATGCGTCCGGCGTAGAGGATCGAGATCTCGATCCCGCGAAATTAATACGAC4020              TCACTATAGGGAGACCACAACGGTTTCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAG4080              GAGATATACCATGGTACCAGACACCGGAAACCCCTGCCACACCACTAAGTTGTTGCACAG4140              AGACTCAGTGGACAGTGCTCCAATCCTCACTGCATTTAACAGCTCACACAAAGGACGGAT4200              TAACTGTAATAGTAACACTACACCCATAGTACATTTAAAAGGTGATGCTAATACTTTAAG4260              ATCTTTAAGATATAGATTTAAAAAGCATTCTACATTGTATACTGCAGTGTCGTCTACATG4320              GCATTGGACAGGACATAATGTAAAACATAAAAGTGCAATTGTTACACTTACATATGATAG4380              TGAATGGCAACGTGACCAATTTTTGTCTCAAGTTAAAATACCAAAAACTATTACAGTGTC4440              TACTGGATTTATGTCTATATGAGGATCCGGCTGCTAACAAAGCCCGAAAGGAAGCTGAGT4500              TGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCT4560              TGAGGGGTTTTTTGCTGAAAGGAGGAACTATATCCGGATATCCACAGGACGGGTGTGGTC4620              GCCATGATCGCGTAGTCGATAGTGGCTCCAAGTAGCGAAGCGAGCAGGACTGGGCGGCGG4680              CCAAAGCGGTCGGACAGTGCTCCGAGAACGGGTGCGCATAGAAATTGCATCAACGCATAT4740              AGCGCTAGCAGCACGCCATAGTGACTGGCGATGCTGTCGGAATGGACGATATCCCGCAAG4800              AGGCCCGGCAGTACCGGCATAACCAAGCCTATGCCTACAGCATCCAGGGTGACGGTGCCG4860              AGGATGACGATGAGCGCATTGTTAGATTTCATACACGGTGCCTGACTGCGTTAGCAATTT4920              AACTGTGATAAACTACCGCATTAAAGCTTATCGATGATAAGCTGTCAAACATGAGAA4977                 (2) INFORMATION FOR SEQ ID NO:18:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 59 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                      CGACACTGCAGTATACAATGTAGAATGCTTTTTAAATCTATATCTTAAAGATCTTAAAG59                 (2) INFORMATION FOR SEQ ID NO:19:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 26 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                      GCGTCGGCCGCCATGCCGGCGATAAT26                                                  (2) INFORMATION FOR SEQ ID NO:20:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 4819 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                      TTCTTGAAGACGAAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGATAAT60                AATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTG120               TTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAAT180               GCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTAT240               TCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGT300               AAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAG360               CGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAA420               AGTTCTGCTATGTGGCGCGGTATTATCCCGTGTTGACGCCGGGCAAGAGCAACTCGGTCG480               CCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCT540               TACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACAC600               TGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCA660               CAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCAT720               ACCAAACGACGAGCGTGACACCACGATGCCTGCAGCAATGGCAACAACGTTGCGCAAACT780               ATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGC840               GGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGA900               TAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGG960               TAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACG1020              AAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCA1080              AGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTA1140              GGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCA1200              CTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCG1260              CGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGA1320              TCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAA1380              TACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCC1440              TACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTG1500              TCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAAC1560              GGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCT1620              ACAGCGTGAGCATTGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCC1680              GGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTG1740              GTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATG1800              CTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCT1860              GGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGA1920              TAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCG1980              CAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTTCTCCTTACGCA2040              TCTGTGCGGTATTTCACACCGCATATATGGTGCACTCTCAGTACAATCTGCTCTGATGCC2100              GCATAGTTAAGCCAGTATACACTCCGCTATCGCTACGTGACTGGGTCATGGCTGCGCCCC2160              GACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTT2220              ACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCAC2280              CGAAACGCGCGAGGCAGCTGCGGTAAAGCTCATCAGCGTGGTCGTGAAGCGATTCACAGA2340              TGTCTGCCTGTTCATCCGCGTCCAGCTCGTTGAGTTTCTCCAGAAGCGTTAATGTCTGGC2400              TTCTGATAAAGCGGGCCATGTTAAGGGCGGTTTTTTCCTGTTTGGTCACTTGATGCCTCC2460              GTGTAAGGGGGAATTTCTGTTCATGGGGGTAATGATACCGATGAAACGAGAGAGGATGCT2520              CACGATACGGGTTACTGATGATGAACATGCCCGGTTACTGGAACGTTGTGAGGGTAAACA2580              ACTGGCGGTATGGATGCGGCGGGACCAGAGAAAAATCACTCAGGGTCAATGCCAGCGCTT2640              CGTTAATACAGATGTAGGTGTTCCACAGGGTAGCCAGCAGCATCCTGCGATGCAGATCCG2700              GAACATAATGGTGCAGGGCGCTGACTTCCGCGTTTCCAGACTTTACGAAACACGGAAACC2760              GAAGACCATTCATGTTGTTGCTCAGGTCGCAGACGTTTTGCAGCAGCAGTCGCTTCACGT2820              TCGCTCGCGTATCGGTGATTCATTCTGCTAACCAGTAAGGCAACCCCGCCAGCCTAGCCG2880              GGTCCTCAACGACAGGAGCACGATCATGCGCACCCGTGGCCAGGACCCAACGCTGCCCGA2940              GATGCGCCGCGTGCGGCTGCTGGAGATGGCGGACGCGATGGATATGTTCTGCCAAGGGTT3000              GGTTTGCGCATTCACAGTTCTCCGCAAGAATTGATTGGCTCCAATTCTTGGAGTGGTGAA3060              TCCGTTAGCGAGGTGCCGCCGGCTTCCATTCAGGTCGAGGTGGCCCGGCTCCATGCACCG3120              CGACGCAACGCGGGGAGGCAGACAAGGTATAGGGCGGCGCCTACAATCCATGCCAACCCG3180              TTCCATGTGCTCGCCGAGGCGGCATAAATCGCCGTGACGATCAGCGGTCCAGTGATCGAA3240              GTTAGGCTGGTAAGAGCCGCGAGCGATCCTTGAAGCTGTCCCTGATGGTCGTCATCTACC3300              TGCCTGGACAGCATGGCCTGCAACGCGGGCATCCCGATGCCGCCGGAAGCGAGAAGAATC3360              ATAATGGGGAAGGCCATCCAGCCTCGCGTCGCGAACGCCAGCAAGACGTAGCCCAGCGCG3420              TCGGCCGCCATGCCGGCGATAATGGCCTGCTTCTCGCCGAAACGTTTGGTGGCGGGACCA3480              GTGACGAAGGCTTGAGCGAGGGCGTGCAAGATTCCGAATACCGCAAGCGACAGGCCGATC3540              ATCGTCGCGCTCCAGCGAAAGCGGTCCTCGCCGAAAATGACCCAGAGCGCTGCCGGCACC3600              TGTCCTACGAGTTGCATGATAAAGAAGACAGTCATAAGTGCGGCGACGATAGTCATGCCC3660              CGCGCCCACCGGAAGGAGCTGACTGGGTTGAAGGCTCTCAAGGGCATCGGTCGACGCTCT3720              CCCTTATGCGACTCCTGCATTAGGAAGCAGCCCAGTAGTAGGTTGAGGCCGTTGAGCACC3780              GCCGCCGCAAGGAATGGTGCATGCAAGGAGATGGCGCCCAACAGTCCCCCGGCCACGGGG3840              CCTGCCACCATACCCACGCCGAAACAAGCGCTCATGAGCCCGAAGTGGCGAGCCCGATCT3900              TCCCCATCGGTGATGTCGGCGATATAGGCGCCAGCAACCGCACCTGTGGCGCCGGTGATG3960              CCGGCCACGATGCGTCCGGCGTAGAGGATCGAGATCTCGATCCCGCGAAATTAATACGAC4020              TCACTATAGGGAGACCACAACGGTTTCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAG4080              GAGATATACATATGGAACCGGTCGACCCGCGTCTGGAACCATGGAAACACCCCGGGTCCC4140              AGCCGAAAACCGCGTTCATCACCAAAGCCCTAGGTATCTCTTACGGCCGTAAAAAACGTC4200              GTCAGCGACGTCGTCCGCCGCAGGGATCCCAGACCCACCAGGTTTCTCTGTCTAAACAGT4260              GATCAGCATTGGCTAGCATGACTGGTGGACAGCAAATGGGTCGCGGATCCGGCTGCTAAC4320              AAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCC4380              CTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGAACTATATCCGGA4440              TATCCACAGGACGGGTGTGGTCGCCATGATCGCGTAGTCGATAGTGGCTCCAAGTAGCGA4500              AGCGAGCAGGACTGGGCGGCGGCCAAAGCGGTCGGACAGTGCTCCGAGAACGGGTGCGCA4560              TAGAAATTGCATCAACGCATATAGCGCTAGCAGCACGCCATAGTGACTGGCGATGCTGTC4620              GGAATGGACGATATCCCGCAAGAGGCCCGGCAGTACCGGCATAACCAAGCCTATGCCTAC4680              AGCATCCAGGGTGACGGTGCCGAGGATGACGATGAGCGCATTGTTAGATTTCATACACGG4740              TGCCTGACTGCGTTAGCAATTTAACTGTGATAAACTACCGCATTAAAGCTTATCGATGAT4800              AAGCTGTCAAACATGAGAA4819                                                       (2) INFORMATION FOR SEQ ID NO:21:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 45 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                      TTTACGGCCGTAAGAGATACCTAGGGCTTTGGTGATGAACGCGGT45                               (2) INFORMATION FOR SEQ ID NO:22:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5574 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                                      TTCTTGAAGACGAAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGATAAT60                AATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTG120               TTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAAT180               GCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTAT240               TCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGT300               AAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAG360               CGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAA420               AGTTCTGCTATGTGGCGCGGTATTATCCCGTGTTGACGCCGGGCAAGAGCAACTCGGTCG480               CCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCT540               TACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACAC600               TGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCA660               CAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCAT720               ACCAAACGACGAGCGTGACACCACGATGCCTGCAGCAATGGCAACAACGTTGCGCAAACT780               ATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGC840               GGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGA900               TAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGG960               TAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACG1020              AAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCA1080              AGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTA1140              GGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCA1200              CTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCG1260              CGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGA1320              TCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAA1380              TACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCC1440              TACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTG1500              TCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAAC1560              GGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCT1620              ACAGCGTGAGCATTGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCC1680              GGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTG1740              GTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATG1800              CTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCT1860              GGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGA1920              TAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCG1980              CAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTTCTCCTTACGCA2040              TCTGTGCGGTATTTCACACCGCATATATGGTGCACTCTCAGTACAATCTGCTCTGATGCC2100              GCATAGTTAAGCCAGTATACACTCCGCTATCGCTACGTGACTGGGTCATGGCTGCGCCCC2160              GACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTT2220              ACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCAC2280              CGAAACGCGCGAGGCAGCTGCGGTAAAGCTCATCAGCGTGGTCGTGAAGCGATTCACAGA2340              TGTCTGCCTGTTCATCCGCGTCCAGCTCGTTGAGTTTCTCCAGAAGCGTTAATGTCTGGC2400              TTCTGATAAAGCGGGCCATGTTAAGGGCGGTTTTTTCCTGTTTGGTCACTTGATGCCTCC2460              GTGTAAGGGGGAATTTCTGTTCATGGGGGTAATGATACCGATGAAACGAGAGAGGATGCT2520              CACGATACGGGTTACTGATGATGAACATGCCCGGTTACTGGAACGTTGTGAGGGTAAACA2580              ACTGGCGGTATGGATGCGGCGGGACCAGAGAAAAATCACTCAGGGTCAATGCCAGCGCTT2640              CGTTAATACAGATGTAGGTGTTCCACAGGGTAGCCAGCAGCATCCTGCGATGCAGATCCG2700              GAACATAATGGTGCAGGGCGCTGACTTCCGCGTTTCCAGACTTTACGAAACACGGAAACC2760              GAAGACCATTCATGTTGTTGCTCAGGTCGCAGACGTTTTGCAGCAGCAGTCGCTTCACGT2820              TCGCTCGCGTATCGGTGATTCATTCTGCTAACCAGTAAGGCAACCCCGCCAGCCTAGCCG2880              GGTCCTCAACGACAGGAGCACGATCATGCGCACCCGTGGCCAGGACCCAACGCTGCCCGA2940              GATGCGCCGCGTGCGGCTGCTGGAGATGGCGGACGCGATGGATATGTTCTGCCAAGGGTT3000              GGTTTGCGCATTCACAGTTCTCCGCAAGAATTGATTGGCTCCAATTCTTGGAGTGGTGAA3060              TCCGTTAGCGAGGTGCCGCCGGCTTCCATTCAGGTCGAGGTGGCCCGGCTCCATGCACCG3120              CGACGCAACGCGGGGAGGCAGACAAGGTATAGGGCGGCGCCTACAATCCATGCCAACCCG3180              TTCCATGTGCTCGCCGAGGCGGCATAAATCGCCGTGACGATCAGCGGTCCAGTGATCGAA3240              GTTAGGCTGGTAAGAGCCGCGAGCGATCCTTGAAGCTGTCCCTGATGGTCGTCATCTACC3300              TGCCTGGACAGCATGGCCTGCAACGCGGGCATCCCGATGCCGCCGGAAGCGAGAAGAATC3360              ATAATGGGGAAGGCCATCCAGCCTCGCGTCGCGAACGCCAGCAAGACGTAGCCCAGCGCG3420              TCGGCCGCCATGCCGGCGATAATGGCCTGCTTCTCGCCGAAACGTTTGGTGGCGGGACCA3480              GTGACGAAGGCTTGAGCGAGGGCGTGCAAGATTCCGAATACCGCAAGCGACAGGCCGATC3540              ATCGTCGCGCTCCAGCGAAAGCGGTCCTCGCCGAAAATGACCCAGAGCGCTGCCGGCACC3600              TGTCCTACGAGTTGCATGATAAAGAAGACAGTCATAAGTGCGGCGACGATAGTCATGCCC3660              CGCGCCCACCGGAAGGAGCTGACTGGGTTGAAGGCTCTCAAGGGCATCGGTCGACGCTCT3720              CCCTTATGCGACTCCTGCATTAGGAAGCAGCCCAGTAGTAGGTTGAGGCCGTTGAGCACC3780              GCCGCCGCAAGGAATGGTGCATGCAAGGAGATGGCGCCCAACAGTCCCCCGGCCACGGGG3840              CCTGCCACCATACCCACGCCGAAACAAGCGCTCATGAGCCCGAAGTGGCGAGCCCGATCT3900              TCCCCATCGGTGATGTCGGCGATATAGGCGCCAGCAACCGCACCTGTGGCGCCGGTGATG3960              CCGGCCACGATGCGTCCGGCGTAGAGGATCGAGATCTCGATCCCGCGAAATTAATACGAC4020              TCACTATAGGGAGACCACAACGGTTTCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAG4080              GAGATATACATATGGAACCGGTCGACCCGCGTCTGGAACCATGGAAACACCCCGGGTCCC4140              AGCCGAAAACCGCGTTCATCACCAAAGCCCTAGGTATCTCTTACGGCCGTAAAAAACGTC4200              GTCAGCGACGTCGTCCGCCGCAGGGATCTTCCATGGCCGGTGCTGGACGCATTTACTATT4260              CTCGCTTTGGTGACGAGGCAGCCAGATTTAGTACAACAGGGCATTACTCTGTAAGAGATC4320              AGGACAGAGTGTATGCTGGTGTCTCATCCACCTCTTCTGATTTTAGAGATCGCCCAGACG4380              GAGTCTGGGTCGCATCCGAAGGACCTGAAGGAGACCCTGCAGGAAAAGAAGCCGAGCCAG4440              CCCAGCCTGTCTCTTCTTTGCTCGGCTCCCCCGCCTGCGGTCCCATCAGAGCAGGCCTCG4500              GTTGGGTACGGGACGGTCCTCGCTCGCACCCCTACAATTTTCCTGCAGGCTCGGGGGGCT4560              CTATTCTCCGCTCTTCCTCCACCCCGGTGCAGGGCACGGTACCGGTGGACTTGGCATCAA4620              GGCAGGAAGAAGAGGAGCAGTCGCCCGACTCCACAGAGGAAGAACCAGTGACTCTCCCAA4680              GGCGCACCACCAATGATGGATTCCACCTGTTAAAGGCAGGAGGGTCATGCTTTGCTCTAA4740              TTTCAGGAACTGCTAACCAGGTAAAGTGCTATCGCTTTCGGGTGAAAAAGAACCATAGAC4800              ATCGCTACGAGAACTGCACCACCACCTGGTTCACAGTTGCTGACAACGGTGCTGAAAGAC4860              AAGGACAAGCACAAATACTGATCACCTTTGGATCGCCAAGTCAAAGGCAAGACTTTCTGA4920              AACATGTACCACTACCTCCTGGAATGAACATTTCCGGCTTTACAGCCAGCTTGGACTTCT4980              GATCACTGCCATTGCCTTTTCTTCATCTGACTGGTGTACTATGCCAAATCTATGGTTTCT5040              ATTGTTCTTGGGACTAGGAAGATCCGGCTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGG5100              CTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGA5160              GGGGTTTTTTGCTGAAAGGAGGAACTATATCCGGATATCCACAGGACGGGTGTGGTCGCC5220              ATGATCGCGTAGTCGATAGTGGCTCCAAGTAGCGAAGCGAGCAGGACTGGGCGGCGGCCA5280              AAGCGGTCGGACAGTGCTCCGAGAACGGGTGCGCATAGAAATTGCATCAACGCATATAGC5340              GCTAGCAGCACGCCATAGTGACTGGCGATGCTGTCGGAATGGACGATATCCCGCAAGAGG5400              CCCGGCAGTACCGGCATAACCAAGCCTATGCCTACAGCATCCAGGGTGACGGTGCCGAGG5460              ATGACGATGAGCGCATTGTTAGATTTCATACACGGTGCCTGACTGCGTTAGCAATTTAAC5520              TGTGATAAACTACCGCATTAAAGCTTATCGATGATAAGCTGTCAAACATGAGAA5574                    (2) INFORMATION FOR SEQ ID NO:23:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                                      GATCCCAGACCCACCAGGTT20                                                        (2) INFORMATION FOR SEQ ID NO:24:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                                      GAACCTGGTGGGTCTGG17                                                           (2) INFORMATION FOR SEQ ID NO:25:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 50 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                                      CGTCCGCCGCAGGGATCGCAGACCCACCAGGTTTCTCTGTCTAAACAGGC50                          (2) INFORMATION FOR SEQ ID NO:26:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 58 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                                      CATGGCCTGTTTAGACAGAGAAACCTGGTGGGTCTGCGATCCCTGCGGCGGACGACGT58                  (2) INFORMATION FOR SEQ ID NO:27:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 48 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                                      CATGTACGGCCGTAAAAAACGTCGTCAGCGACGTCGTCCGCCGGACAC48                            (2) INFORMATION FOR SEQ ID NO:28:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 46 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:                                      CGGTGTCCGGCGGACGACGTCGCTGACGACGTTTTTTACGGCCGTA46                              (2) INFORMATION FOR SEQ ID NO:29:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 26 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:                                      ATCATCGATAAGCTTTAATGCGGTAG26                                                  (2) INFORMATION FOR SEQ ID NO:30:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 52 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:                                      ACTTTAAGAAGGAGATATACATATGTTCATCACCAAAGCCCTAGGTATCTCT52                        (2) INFORMATION FOR SEQ ID NO:31:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 51 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:                                      ACTTTAAGAAGGAGATATACATATGTACGGCCGTAAAAAACGTCGTCAGCG51                         (2) INFORMATION FOR SEQ ID NO:32:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 52 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:                                      AACGTCGTCAGCGACGTCGTCCGCCGGACACCGGAAACCCCTGCCACACCAC52                        (2) INFORMATION FOR SEQ ID NO:33:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:                                      CGAAAAGTGCCACCTGACGTCTAAGAAACC30                                              (2) INFORMATION FOR SEQ ID NO:34:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 26 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:                                      CTCCCATGGCTAGCAACACTACACCC26                                                  (2) INFORMATION FOR SEQ ID NO:35:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:                                      GAAGATCTTC10                                                                  (2) INFORMATION FOR SEQ ID NO:36:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:                                      CAGAGGAAGCCATGGTGACTCTCCCAA27                                                 (2) INFORMATION FOR SEQ ID NO:37:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:                                      AAGGCAATGGATCCGATCAGAAGTCCA27                                                 (2) INFORMATION FOR SEQ ID NO:38:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 134 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:                                      MetTyrGlyArgLysLysArgArgGlnArgArgArgProProAspThr                              151015                                                                        GlyAsnProCysHisThrThrLysLeuLeuHisArgAspSerValAsp                              202530                                                                        SerAlaProIleLeuThrAlaPheAsnSerSerHisLysGlyArgIle                              354045                                                                        AsnCysAsnSerAsnThrThrProIleValHisLeuLysGlyAspAla                              505560                                                                        AsnThrLeuLysCysLeuArgTyrArgPheLysLysHisCysThrLeu                              65707580                                                                      TyrThrAlaValSerSerThrTrpHisTrpThrGlyHisAsnValLys                              859095                                                                        HisLysSerAlaIleValThrLeuThrTyrAspSerGluTrpGlnArg                              100105110                                                                     AspGlnPheLeuSerGlnValLysIleProLysThrIleThrValSer                              115120125                                                                     ThrGlyPheMetSerIle                                                            130                                                                           (2) INFORMATION FOR SEQ ID NO:39:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 55 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:                                      CATGTACGGCCGTAAAAAACGTCGTCAGCGACGTCGTCCGCTGAGTCAGGCCCAG55                     (2) INFORMATION FOR SEQ ID NO:40:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 51 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:                                      CTGGGCCTGACTCAGCGGACGACGTCGCTGACGACGTTTTTTACGGCCGTA51                         (2) INFORMATION FOR SEQ ID NO:41:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 46 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:                                      TCCTTCCTGTCCGCTGGTCAGCGCCCGCGCCGCCTGTCCACCTAAG46                              (2) INFORMATION FOR SEQ ID NO:42:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 54 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:                                      AATTCTTAGGTGGACAGGCGGCGCGGGCGCTGACCAGCGGACAGGAAGGACATG54                      (2) INFORMATION FOR SEQ ID NO:43:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 39 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:                                      GGGGACTTTCCGCTGGGGACTTTCCACGGGGGACTTTCC39                                     (2) INFORMATION FOR SEQ ID NO:44:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 39 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:                                      GGAAAGTCCCCCGTGGAAAGTCCCCAGCGGAAAGTCCCC39                                     (2) INFORMATION FOR SEQ ID NO:45:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 39 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:                                      GTCTACTTTCCGCTGTCTACTTTCCACGGTCTACTTTCC39                                     (2) INFORMATION FOR SEQ ID NO:46:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 39 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:                                      GGAAAGTAGACCGTGGAAAGTAGACAGCGGAAAGTAGAC39                                     (2) INFORMATION FOR SEQ ID NO:47:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 12 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:                                      TyrGlyArgLysLysArgArgGlnArgArgArgPro                                          1510                                                                          (2) INFORMATION FOR SEQ ID NO:48:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 26 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:                                      TyrGlyArgLysLysArgArgGlnArgArgArgProProGlnGlySer                              151015                                                                        GlnThrHisGlnValSerLeuSerLysGln                                                2025                                                                          (2) INFORMATION FOR SEQ ID NO:49:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 35 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:49:                                      PheIleThrLysAlaLeuGlyIleSerTyrGlyArgLysLysArgArg                              151015                                                                        GlnArgArgArgProProGlnGlySerGlnThrHisGlnValSerLeu                              202530                                                                        SerLysGln                                                                     35                                                                            (2) INFORMATION FOR SEQ ID NO:50:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:50:                                      PheIleThrLysAlaLeuGlyIleSerTyrGlyArgLysLysArgArg                              151015                                                                        GlnArgArgArgPro                                                               20                                                                            (2) INFORMATION FOR SEQ ID NO:51:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 121 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:51:                                      ProAspThrGlyAsnProCysHisThrThrLysLeuLeuHisArgAsp                              151015                                                                        SerValAspSerAlaProIleLeuThrAlaPheAsnSerSerHisLys                              202530                                                                        GlyArgIleAsnCysAsnSerAsnThrThrProIleValHisLeuLys                              354045                                                                        GlyAspAlaAsnThrLeuLysCysLeuArgTyrArgPheLysLysHis                              505560                                                                        CysThrLeuTyrThrAlaValSerSerThrTrpHisTrpThrGlyHis                              65707580                                                                      AsnValLysHisLysSerAlaIleValThrLeuThrTyrAspSerGlu                              859095                                                                        TrpGlnArgAspGlnPheLeuSerGlnValLysIleProLysThrIle                              100105110                                                                     ThrValSerThrGlyPheMetSerIle                                                   115120                                                                        (2) INFORMATION FOR SEQ ID NO:52:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:52:                                      GlyArgLysLysArgArgGlnArgArgArgProProGlnGlySer                                 151015                                                                        (2) INFORMATION FOR SEQ ID NO:53:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:53:                                      PheIleThrLysAlaLeuGlyIleSerTyrGlyArgLysLysArgArg                              151015                                                                        GlnArgArgArgProProGlnGlySer                                                   2025                                                                          (2) INFORMATION FOR SEQ ID NO:54:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 85 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:54:                                      CysAsnSerAsnThrThrProIleValHisLeuLysGlyAspAlaAsn                              151015                                                                        ThrLeuLysCysLeuArgTyrArgPheLysLysHisCysThrLeuTyr                              202530                                                                        ThrAlaValSerSerThrTrpHisTrpThrGlyHisAsnValLysHis                              354045                                                                        LysSerAlaIleValThrLeuThrTyrAspSerGluTrpGlnArgAsp                              505560                                                                        GlnPheLeuSerGlnValLysIleProLysThrIleThrValSerThr                              65707580                                                                      GlyPheMetSerIle                                                               85                                                                            (2) INFORMATION FOR SEQ ID NO:55:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 121 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:55:                                      ProAspThrGlyAsnProCysHisThrThrLysLeuLeuHisArgAsp                              151015                                                                        SerValAspSerAlaProIleLeuThrAlaPheAsnSerSerHisLys                              202530                                                                        GlyArgIleAsnCysAsnSerAsnThrThrProIleValHisLeuLys                              354045                                                                        GlyAspAlaAsnThrLeuLysSerLeuArgTyrArgPheLysLysHis                              505560                                                                        SerThrLeuTyrThrAlaValSerSerThrTrpHisTrpThrGlyHis                              65707580                                                                      AsnValLysHisLysSerAlaIleValThrLeuThrTyrAspSerGlu                              859095                                                                        TrpGlnArgAspGlnPheLeuSerGlnValLysIleProLysThrIle                              100105110                                                                     ThrValSerThrGlyPheMetSerIle                                                   115120                                                                        (2) INFORMATION FOR SEQ ID NO:56:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 161 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:56:                                      LeuGlyTrpValArgAspGlyProArgSerHisProTyrAsnPhePro                              151015                                                                        AlaGlySerGlyGlySerIleLeuArgSerSerSerThrProValGln                              202530                                                                        GlyThrValProValAspLeuAlaSerArgGlnGluGluGluGluGln                              354045                                                                        SerProAspSerThrGluGluGluProValThrLeuProArgArgThr                              505560                                                                        ThrAsnAspGlyPheHisLeuLeuLysAlaGlyGlySerCysPheAla                              65707580                                                                      LeuIleSerGlyThrAlaAsnGlnValLysCysTyrArgPheArgVal                              859095                                                                        LysLysAsnHisArgHisArgTyrGluAsnCysThrThrThrTrpPhe                              100105110                                                                     ThrValAlaAspAsnGlyAlaGluArgGlnGlyGlnAlaGlnIleLeu                              115120125                                                                     IleThrPheGlySerProSerGlnArgGlnAspPheLeuLysHisVal                              130135140                                                                     ProLeuProProGlyMetAsnIleSerGlyPheThrAlaSerLeuAsp                              145150155160                                                                  Phe                                                                           (2) INFORMATION FOR SEQ ID NO:57:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 249 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:57:                                      MetAlaGlyAlaGlyArgIleTyrTyrSerArgPheGlyAspGluAla                              151015                                                                        AlaArgPheSerThrThrGlyHisTyrSerValArgAspGlnAspArg                              202530                                                                        ValTyrAlaGlyValSerSerThrSerSerAspPheArgAspArgPro                              354045                                                                        AspGlyValTrpValAlaSerGluGlyProGluGlyAspProAlaGly                              505560                                                                        LysGluAlaGluProAlaGlnProValSerSerLeuLeuGlySerPro                              65707580                                                                      AlaCysGlyProIleArgAlaGlyLeuGlyTrpValArgAspGlyPro                              859095                                                                        ArgSerHisProTyrAsnPheProAlaGlySerGlyGlySerIleLeu                              100105110                                                                     ArgSerSerSerThrProValGlnGlyThrValProValAspLeuAla                              115120125                                                                     SerArgGlnGluGluGluGluGlnSerProAspSerThrGluGluGlu                              130135140                                                                     ProValThrLeuProArgArgThrThrAsnAspGlyPheHisLeuLeu                              145150155160                                                                  LysAlaGlyGlySerCysPheAlaLeuIleSerGlyThrAlaAsnGln                              165170175                                                                     ValLysCysTyrArgPheArgValLysLysAsnHisArgHisArgTyr                              180185190                                                                     GluAsnCysThrThrThrTrpPheThrValAlaAspAsnGlyAlaGlu                              195200205                                                                     ArgGlnGlyGlnAlaGlnIleLeuIleThrPheGlySerProSerGln                              210215220                                                                     ArgGlnAspPheLeuLysHisValProLeuProProGlyMetAsnIle                              225230235240                                                                  SerGlyPheThrAlaSerLeuAspPhe                                                   245                                                                           (2) INFORMATION FOR SEQ ID NO:58:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 385 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:58:                                      MetTyrGlyArgLysLysArgArgGlnArgArgArgProLeuSerGln                              151015                                                                        AlaGlnLeuMetProSerProProMetProValProProAlaAlaLeu                              202530                                                                        PheAsnArgLeuLeuAspAspLeuGlyPheSerAlaGlyProAlaLeu                              354045                                                                        CysThrMetLeuAspThrTrpAsnGluAspLeuPheSerGlyPhePro                              505560                                                                        ThrAsnAlaAspMetTyrArgGluCysLysPheLeuSerThrLeuPro                              65707580                                                                      SerAspValIleAspTrpGlyAspAlaHisValProGluArgSerPro                              859095                                                                        IleAspIleArgAlaHisGlyAspValAlaPheProThrLeuProAla                              100105110                                                                     ThrArgAspGluLeuProSerTyrTyrGluAlaMetAlaGlnPhePhe                              115120125                                                                     ArgGlyGluLeuArgAlaArgGluGluSerTyrArgThrValLeuAla                              130135140                                                                     AsnPheCysSerAlaLeuTyrArgTyrLeuArgAlaSerValArgGln                              145150155160                                                                  LeuHisArgGlnAlaHisMetArgGlyArgAsnArgAspLeuArgGlu                              165170175                                                                     MetLeuArgThrThrIleAlaAspArgTyrTyrArgGluThrAlaArg                              180185190                                                                     LeuAlaArgValLeuPheLeuHisLeuTyrLeuPheLeuSerArgGlu                              195200205                                                                     IleLeuTrpAlaAlaTyrAlaGluGlnMetMetArgProAspLeuPhe                              210215220                                                                     AspGlyLeuCysCysAspLeuGluSerTrpArgGlnLeuAlaCysLeu                              225230235240                                                                  PheGlnProLeuMetPheIleAsnGlySerLeuThrValArgGlyVal                              245250255                                                                     ProValGluAlaArgArgLeuArgGluLeuAsnHisIleArgGluHis                              260265270                                                                     LeuAsnLeuProLeuValArgSerAlaAlaAlaGluGluProGlyAla                              275280285                                                                     ProLeuThrThrProProValLeuGlnGlyAsnGlnAlaArgSerSer                              290295300                                                                     GlyTyrPheMetLeuLeuIleArgAlaLysLeuAspSerTyrSerSer                              305310315320                                                                  ValAlaThrSerGluGlyGluSerValMetArgGluHisAlaTyrSer                              325330335                                                                     ArgGlyArgThrArgAsnAsnTyrGlySerThrIleGluGlyLeuLeu                              340345350                                                                     AspLeuProAspAspAspAspAlaProAlaGluAlaGlyLeuValAla                              355360365                                                                     ProArgMetSerPheLeuSerAlaGlyGlnArgProArgArgLeuSer                              370375380                                                                     Thr                                                                           385                                                                           (2) INFORMATION FOR SEQ ID NO:59:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 148 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:59:                                      MetTyrGlyArgLysLysArgArgGlnArgArgArgProProGlnGly                              151015                                                                        SerGlnThrHisGlnValSerLeuSerLysGlnProAspThrGlyAsn                              202530                                                                        ProCysHisThrThrLysLeuLeuHisArgAspSerValAspSerAla                              354045                                                                        ProIleLeuThrAlaPheAsnSerSerHisLysGlyArgIleAsnCys                              505560                                                                        AsnSerAsnThrThrProIleValHisLeuLysGlyAspAlaAsnThr                              65707580                                                                      LeuLysCysLeuArgTyrArgPheLysLysHisCysThrLeuTyrThr                              859095                                                                        AlaValSerSerThrTrpHisTrpThrGlyHisAsnValLysHisLys                              100105110                                                                     SerAlaIleValThrLeuThrTyrAspSerGluTrpGlnArgAspGln                              115120125                                                                     PheLeuSerGlnValLysIleProLysThrIleThrValSerThrGly                              130135140                                                                     PheMetSerIle                                                                  145                                                                           (2) INFORMATION FOR SEQ ID NO:60:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 157 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:60:                                      MetPheIleThrLysAlaLeuGlyIleSerTyrGlyArgLysLysArg                              151015                                                                        ArgGlnArgArgArgProProGlnGlySerGlnThrHisGlnValSer                              202530                                                                        LeuSerLysGlnProAspThrGlyAsnProCysHisThrThrLysLeu                              354045                                                                        LeuHisArgAspSerValAspSerAlaProIleLeuThrAlaPheAsn                              505560                                                                        SerSerHisLysGlyArgIleAsnCysAsnSerAsnThrThrProIle                              65707580                                                                      ValHisLeuLysGlyAspAlaAsnThrLeuLysCysLeuArgTyrArg                              859095                                                                        PheLysLysHisCysThrLeuTyrThrAlaValSerSerThrTrpHis                              100105110                                                                     TrpThrGlyHisAsnValLysHisLysSerAlaIleValThrLeuThr                              115120125                                                                     TyrAspSerGluTrpGlnArgAspGlnPheLeuSerGlnValLysIle                              130135140                                                                     ProLysThrIleThrValSerThrGlyPheMetSerIle                                       145150155                                                                     (2) INFORMATION FOR SEQ ID NO:61:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 177 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:61:                                      MetGlyArgLysLysArgArgGlnArgArgArgProProGlnGlySer                              151015                                                                        LeuGlyTrpValArgAspGlyProArgSerHisProTyrAsnPhePro                              202530                                                                        AlaGlySerGlyGlySerIleLeuArgSerSerSerThrProValGln                              354045                                                                        GlyThrValProValAspLeuAlaSerArgGlnGluGluGluGluGln                              505560                                                                        SerProAspSerThrGluGluGluProValThrLeuProArgArgThr                              65707580                                                                      ThrAsnAspGlyPheHisLeuLeuLysAlaGlyGlySerCysPheAla                              859095                                                                        LeuIleSerGlyThrAlaAsnGlnValLysCysTyrArgPheArgVal                              100105110                                                                     LysLysAsnHisArgHisArgTyrGluAsnCysThrThrThrTrpPhe                              115120125                                                                     ThrValAlaAspAsnGlyAlaGluArgGlnGlyGlnAlaGlnIleLeu                              130135140                                                                     IleThrPheGlySerProSerGlnArgGlnAspPheLeuLysHisVal                              145150155160                                                                  ProLeuProProGlyMetAsnIleSerGlyPheThrAlaSerLeuAsp                              165170175                                                                     Phe                                                                           (2) INFORMATION FOR SEQ ID NO:62:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 187 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:62:                                      MetPheIleThrLysAlaLeuGlyIleSerTyrGlyArgLysLysArg                              151015                                                                        ArgGlnArgArgArgProProGlnGlySerLeuGlyTrpValArgAsp                              202530                                                                        GlyProArgSerHisProTyrAsnPheProAlaGlySerGlyGlySer                              354045                                                                        IleLeuArgSerSerSerThrProValGlnGlyThrValProValAsp                              505560                                                                        LeuAlaSerArgGlnGluGluGluGluGlnSerProAspSerThrGlu                              65707580                                                                      GluGluProValThrLeuProArgArgThrThrAsnAspGlyPheHis                              859095                                                                        LeuLeuLysAlaGlyGlySerCysPheAlaLeuIleSerGlyThrAla                              100105110                                                                     AsnGlnValLysCysTyrArgPheArgValLysLysAsnHisArgHis                              115120125                                                                     ArgTyrGluAsnCysThrThrThrTrpPheThrValAlaAspAsnGly                              130135140                                                                     AlaGluArgGlnGlyGlnAlaGlnIleLeuIleThrPheGlySerPro                              145150155160                                                                  SerGlnArgGlnAspPheLeuLysHisValProLeuProProGlyMet                              165170175                                                                     AsnIleSerGlyPheThrAlaSerLeuAspPhe                                             180185                                                                        (2) INFORMATION FOR SEQ ID NO:63:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 143 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:63:                                      MetPheIleThrLysAlaLeuGlyIleSerTyrGlyArgLysLysArg                              151015                                                                        ArgGlnArgArgArgProProAspThrGlyAsnProCysHisThrThr                              202530                                                                        LysLeuLeuHisArgAspSerValAspSerAlaProIleLeuThrAla                              354045                                                                        PheAsnSerSerHisLysGlyArgIleAsnCysAsnSerAsnThrThr                              505560                                                                        ProIleValHisLeuLysGlyAspAlaAsnThrLeuLysCysLeuArg                              65707580                                                                      TyrArgPheLysLysHisCysThrLeuTyrThrAlaValSerSerThr                              859095                                                                        TrpHisTrpThrGlyHisAsnValLysHisLysSerAlaIleValThr                              100105110                                                                     LeuThrTyrAspSerGluTrpGlnArgAspGlnPheLeuSerGlnVal                              115120125                                                                     LysIleProLysThrIleThrValSerThrGlyPheMetSerIle                                 130135140                                                                     (2) INFORMATION FOR SEQ ID NO:64:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 46 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:64:                                      CGTCCGCCGCAGGGATCCCAGACCCACCAGGTTCCGGTTACTCTGC46                              (2) INFORMATION FOR SEQ ID NO:65:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 54 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:65:                                      CTTGGCAGAGTAACCGGAACCTGGTGGGTCTGGGATCCCTGCGGCGGACGACGT54                      (2) INFORMATION FOR SEQ ID NO:66:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:66:                                      CTAGTGGCTCGAGATTCCG19                                                         (2) INFORMATION FOR SEQ ID NO:67:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:67:                                      GATCCGGAATCTCGAGCCA19                                                         (2) INFORMATION FOR SEQ ID NO:68:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:68:                                      CTCGAGAAGCTTGACGGATCCG22                                                      (2) INFORMATION FOR SEQ ID NO:69:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:69:                                      AATTCGGATCCGTCAAGCTTCTCGAGACGT30                                              __________________________________________________________________________

We claim:
 1. An agent for delivery of a cargo moiety consisting of acargo moiety in combination with a transport moiety, wherein thetransport moiety has the following characteristics:(i) the presence ofamino acids 49-57 of HIV tat protein; (ii) the absence of amino acids22-36 of HIV tat protein; and (iii) the absence of amino acids 73-86 ofHIV tat protein.
 2. The agent of claim 1, wherein the cargo moietyretains biological activity following transport moiety-dependentintracellular delivery.
 3. The agent of claim 2, wherein the agent is afusion protein having a carboxy-terminal that is the cargo moiety andhaving an amino-terminal that is the transport moiety.
 4. The agent ofclaim 3, wherein the cargo moiety consists of amino acids 245-365 ofhuman papillomavirus E2 protein (SEQ ID NO: 51).
 5. The agent of claim3, wherein the carboxy-terminal cargo moiety is selected from the groupconsisting of therapeutic molecules, prophylactic molecules anddiagnostic molecules.
 6. The agent of claim 3, wherein the cargo moietyis an E2 repressor consisting of amino acids 250-410 of bovinepapillomavirus E2 protein (SEQ ID NO: 56).
 7. The agent of claim 3,wherein the cargo moiety is selected from the group consisting of:(a)amino acids 245-365 of human papillomavirus E2 protein (SEQ ID NO: 51);and (b) amino acids 245-365 of human papillomavirus E2 protein, whereinamino acids 300 and 309 have been changed to serine (SEQ ID NO: 55). 8.The agent of claim 3, wherein the cargo moiety is an antigen.
 9. Afusion protein comprising a cargo moiety linked to a target moiety,wherein the cargo moiety is a human papillomavirus E2 repressor thatretains its biological activity after delivery into a target cell andwherein the transport moiety is selected from the group consistingof:(a) amino acids 47-58 of HIV tat protein (SEQ ID NO: 47); (b) aminoacids 47-72 of HIV tat protein (SEQ ID NO: 48); (c) amino acids 38-72 ofHIV tat protein (SEQ ID NO: 49); (d) amino acids 38-58 of HIV tatprotein (SEQ ID NO: 50); (e) amino acids 37-58 of HIV tat protein (SEQID NO: 3); (f) amino acids 1-21 and 38-72 of HIV tat protein (SEQ ID NO:7); (g) amino acids 47-62 of HIV tat protein (SEQ ID NO: 52); and (h)amino acids 38-62 of HIV tat protein (SEQ ID NO: 53).
 10. The fusionprotein of claim 9, wherein the transport moiety is preceded by anamino-terminal methionine.
 11. An agent for delivery of a cargo moiety,the agent comprising an HIV tat protein characterized by:(i) thepresence of amino acids 49-57 of HIV tat protein; (ii) the absence ofamino acids 22-36 of HIV tat protein; and (iii) the absence of aminoacids 73-86 of HIV tat protein.
 12. An agent for delivery of a cargomoiety, the agent comprising an HIV tat protein selected from the groupconsisting of:(a) amino acids 47-72 of HIV tat protein (SEQ ID NO: 48);(b) amino acids 38-72 of HIV tat protein (SEQ ID NO: 49); (c) aminoacids 37-58 of HIV tat protein (SEQ ID NO: 3); and (d) amino acids 1-21and 38-72 of HIV tat protein (SEQ ID NO: 7).
 13. A composition of mattercomprising an HIV tat protein characterized by:(i) the presence of aminoacids 49-57 of HIV tat protein; (ii) the absence of amino acids 22-36 ofHIV tat protein; and (iii) the absence of amino acids 73-86 of HIV tatprotein.
 14. A composition of matter comprising an HIV tat proteinselected from the group consisting of:(a) amino acids 47-72 of HIV tatprotein (SEQ ID NO: 48); (b) amino acids 38-72 of HIV tat protein (SEQID NO: 49); (c) amino acids 37-58 of HIV tat protein (SEQ ID NO: 3); and(d) amino acids 1-21 and 38-72 of HIV tat protein (SEQ ID NO: 7).